Crosstalk between E2F3 and p19ARF/p53
in the regulation of cell cycle progression and tumorigenesis
by
Aaron Spencer Aslanian
B.S. Biology, M.S. Biology
Stanford University, 1999
Submitted to the Department of Biology
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
at the
Massachusetts Institute of Technology
September 2005
( 2005 Massachusetts Institute of Technology. All rights reserved.
I
Signature of Author
A.
w
-
IV
/
Aaron S. Aslanian
Department of Biology
Certified by
Jacqueline A. Lees
Thesis Supervisor
Accepted by
MASSCHUSTTSINSTTUT
i
II
MASSACHUSETTS INSTllTE.
OF TECHNOLOGY
AUG 2 9 2005
LIBRARIES
II
5--'
qStpnhn P Rll
Graduate Committee Chairman
ARCHIVES
ABSTRACT
E2F activity was originally identified as a critical component of the cellular
machinery responsible for promoting cell cycle progression that is co-opted during
transformation and tumorigenesis. Classic E2F target genes have functions required for
S-phase entry and progression. However, the role of E2F has expanded in recent years
through the identification of novel E2F target genes. Now E2F is linked to many
different cellular processes beyond basic cell cycle control. One such example is
p19ARF, a positive upstream regulator of the p53 tumor suppressor protein.
This dissertation examines the relationship between one member of the E2F
family of transcription factors, E2F3, and the p19ARF/p53 pathway. E2F3 has
previously been shown to be critical for cell cycle re-entry, cellular proliferation, and
tumor development. The contribution of p19ARF/p53 to E2F3 function was assessed
through the generation of compound mutant cells and mice. The nature of the
relationship between E2F3, p19ARF, and p53 was highly context dependent.
E2F3 is required to repress pl9Arf expression in quiescent cells. This places
E2F3 upstream of p19ARF and p53 during cell cycle re-entry. The loss of either p9Arf
or p53 completely suppresses the cell cycle re-entry defects in E2f3-deficient cells. In
contrast, E2f3-loss impairs the proliferation of both p]9Arf and p53 knockout cells that
are cycling asynchronously.
In this setting, E2F3 appears to function primarily through
pl9Arf-independent mechanisms. Additionally, the impairment of cellular proliferation
in this setting occurs without any detectable defect in classic E2F target gene expression.
Finally, the effect of E2f3-loss in transformation and tumorigenesis appears to depend on
the underlying genetic alterations. In the background of pl9Arf-deficiency, E2f3-loss
impairs tranformation and tumorigenesis. However, in the background of p53deficiency, E2f3-loss has no effect on transformation or tumorigenesis. This distinction
between p19ARF and p53 also suggests that the regulation of cellular proliferation cannot
be the only function of E2F3 relevant to transformation and tumorigenesis.
This work has established that the loss of E2f3 has multiple effects on cells that
are relevant to tumor biology. As these roles become more clearly defined, we will gain
a better understanding of what are the consequences of E2F activity deregulation.
2
ACKNOWLEDGEMENTS
I would like to thank my adviser, Jackie, for her helpful advice over the years. I
would also like to thank the members of my thesis committee, Terry Orr-Weaver, Steve
Bell, Tyler Jacks, David Housman, and Phil Hinds, for the time and thought that they put
into this challenging project. Additionally, I am thankful for the opportunity to work and
interact with the members of my lab, the fifth floor of the Center for Cancer Research,
MIT, and the Boston scientific community.
I am especially indebted to my parents, who have always sought to provide the
best for me. I am continually grateful for their support and caring. Finally, I would like
to dedicate this dissertation to my Grandma Coffee and Grandma Ethel, who were my
best friends as a child, and from whom I learned so much.
3
TABLE OF CONTENTS
C hapter 1: Introduction
-- -- -- -- -- -- -- -- - -- -- ,---- -- --
-- -- -- -
The cell cycle, exit and checkpoints --------------------------------------
6
7
The RB pathway -..
-------------------------10
.......
The E2F family of transcription factors------------------------------------12
Regulation of E2F by the RB pathway------------------------------------23
The p53 pathway ------------------
31
References
46
Chapter 2: Repression of the Arftumor suppressor by E2F3 is required for normal cell
cycle kinetics -----------------------------------77
Abstract
78
Introduction
79
Results
82
Discussion
94
Materials and Methods
References
101
105
Chapter 3: The proliferation defect in cycling E2f3-deficient cells is pl9Arf-independent
110
Summary
--------------------------------
111
Results and Discussion
Materials and Methods
References
112
123
126
Chapter 4: Tumor development in the absence of p53 is not dependent on the regulation
of proliferation by E2f3 ---------------------------------------128
Abstract
Introduction
Results
Discussion .141
Materials and Methods
References
129
130
133
145
147
4
Chapter 5: Discussion ,..
.
...............................
149
S-phase: Cell cycle re-entry versus the proliferation of cycling cells ----------151
E2F3 regulation of the p53 pathway through p]9Arfand its role in proliferation
.-.......................................................
153
E2F3 regulation of E2F target genes and their role in proliferation Dissecting E2F3 function ----------------------------Concluding remarks ..-..........................
..........
160
164
169
References
171
Appendix A: E2f3-loss leads to increased cell size -.................................
References
176
185
Appendix B: E2f3-loss impairs E2F target gene expression in a pure strain background
..........-..................................................
187
References
191
Appendix C: 'The role of E2f3 in tumorigenesis in p53 heterozygous mice -------------192
References
202
Appendix D: E2f3-loss impairs cell cycle exit -.................................
References
203
213
Appendix E: The retinoblastoma protein: will the real tumor suppressor function please
stand up? -,,---------------------------------------------------215
Preface
Introduction
Pocket protein family ---------------------------------
216
216
217
Cell cycle progression ----------------------------Cell cycle exit ---------------------------Differentiation
Conclusions
Text box 1
219
223
225
228
230
Text box 2
References
231
232
5
CHAPTER
1
INTRODUCTION
6
Progression through the mitotic cell cycle requires duplication of the genome and
equal segregation of DNA into two daughter cells. Faithful transmission of genetic
material during cell division is not only essential for proper development, but also
survival. Tumor development is a complex, multi-step process that can adversely affect
an organism's survival (Vogelstein and Kinzler, 1993). Errors in the genome and in gene
regulation that arise during cell divisions can result in the disruption of key pathways
and, ultimately, tumorigenesis (Sherr, 2004; Vogelstein and Kinzler, 2004).
Understanding the checks and balances through which cells normally control these
pathways will improve our ability to treat and perhaps prevent cancers. The regulation of
cellular proliferation represents one critical process targeted by tumor cells (Hanahan and
Weinberg, 2000).
THE CELL CYCLE, EXIT AND CHECKPOINTS
The cell cycle consists of a period of DNA synthesis (S-phase) and a period of
DNA segregation (mitosis) separated by two gap phases (Gi and G2) during which cells
grow and synthesize proteins, preparing themselves for the next phase of the cell cycle.
Concurrent with the cell cycle regulation of DNA synthesis and segregation is the
duplication of the centrosome, an organelle that participates in the equal segregation of
the DNA during mitosis (Hansen et al., 2002; Wang et al., 2004b).
In mammalian cells, the decision to proceed through the cell cycle is made late in
G1, at what has been defined as the restriction point (Pardee, 1974). If the requirements
for cell cycle progression are not met, cells temporarily exit the cell cycle and arrest in a
GO/G1 state known as quiescence. The decision to proceed through the restriction point
7
is dependent on appropriate growth factor signaling (Zetterberg and Larsson, 1985).
Afterward, cells are committed to proceed through the cell cycle and are no longer
dependent on growth factor levels. In actuality, there are probably several "restriction
points" feeding into this decision, as cell-cell contact and cell adhesion are also factors
that affect continued cell cycle progression prior to S-phase (Gad et al., 2004). Quiescent
cells can be stimulated to re-enter the cell cycle by restoring them to the appropriate
growth conditions. Many cell cycle regulatory proteins must be synthesized anew once
quiescent cells re-enter the cell cycle, marking one important distinction between this
process and the proliferation of continuously cycling cells (Coverley et al., 2002;
Petersen et al., 2000; Wirth et al., 2004).
Primary cells do not proliferate indefinitely, but instead possess a finite replicative
lifespan (Hayflick, 1965; Hayflick and Moorhead, 1961). This was first described in
human cells as the Hayflick limit. Cells that have reached the end of their replicative
potential are described as senescent. Senescence is a permanent GO/GI state of cell cycle
exit, although recent work has demonstrated that genetic manipulation can reverse
senescence (Sage et al., 2003). Mutations that arise prior to the onset of senescence can
also confer on cells the ability to bypass senescence. These cells are then immortal. In
human cells, senescence has been shown to occur as a result of telomere attrition (Harley
et al., 1990). Senescence-like phenotypes can be induced artificially, such as through the
generation of reactive oxygen species (Parrinello et al., 2003). Murine cells also undergo
senescence, and the availability of knockout strains has been used to probe the genetic
requirements for senescence in this system (Blasco et al., 1997). The presence of active
telomerase and long telomeres at the onset of senescence in murine cells suggest that the
8
process of senescence differs between human and murine systems. One conclusion that
has been made from these studies is that the occurrence of senescence is merely a tissue
culture phenomenon (Sherr and DePinho, 2000). However, an alternate theory supported
by in vivo data indicates that senescence may be part of a tumor surveillance mechanism
(Chen et al., 2005; Denchi et al., 2005; Zindy et al., 2003).
In an adult organism, most cells are no longer actively cycling. Differentiation
involves the permanent exit from the cell cycle and initiation of a tissue-specific pattern
of gene expression. Indeed, from the point of fertilization, the zygote proceeds from a
pluripotent state and gives rise to a multitude of highly specialized and distinct cell types.
Our understanding of the cell cycle comes primarily from the study of primary fibroblasts
and tumor cell lines. It is likely that the regulation of the cell cycle and other processes
varies subtly among different cell types, and work done in other systems such as
lymphocytes suggests that this is indeed the case (Randle et al., 2001). Additionally,
forced expression of certain pro-proliferative genes has been shown to prevent complete
withdrawal from the cell cycle in differentiated cells (Chen and Lee, 1999; Scheijen et
al., 2003). This may be relevant in our understanding of the development of cancer.
Cell cycle checkpoints exist to remedy errors that occur during DNA replication
and segregation (Bharadwaj and Yu, 2004; Kastan and Bartek, 2004). These checkpoints
are designed to prevent cells from proceeding through the cell cycle when it would
compromise the fidelity of the genome. In accordance with this role, checkpoints often
act through the reversible inactivation of proteins normally involved in promoting cell
cycle progression. It is easy to see how the lack of cell cycle checkpoints could quickly
9
promote the development of cancer through increased mutation rate or genomic
instability.
This thesis examines two key pathways, the RB pathway and p53 pathway, which
regulate S-phase entry and progression (Figure 1). Growth factor stimulated cellular
proliferation signals ultimately feed through the RB pathway, making its components
critical targets of tumorigenesis.
The p53 pathway functions as the guardian of the
genome, regulating S-phase entry and progression in response to cellular stress. As a
result of bearing this great burden, p53 is functionally inactivated in almost all cancers.
There is considerable crosstalk between these two pathways, which represents an
emerging aspect of their function in tumorigenesis. The following sections review the
role and regulation of key components of the RB pathway and p53 pathway.
THE RB PATHWAY
The E2F family of transcription factors represents the key downstream target of
the RB pathway (Trimarchi and Lees, 2002). Classic E2F target genes play an important
role in cellular proliferation through the control of S-phase entry and progression. E2F
activity is regulated primarily through association with the tumor suppressor protein pRB
and the related proteins p107 and p130, which comprise the pocket protein family.
Sequential phosphorylation of the pocket proteins by cyclin dependent kinases (cdks)
modulate the interaction between E2Fs and the pocket proteins. Finally, two families of
cdk inhibitory proteins (the INK4 family and the CIP/KIP family) cooperate with the
pocket proteins to enforce both temporary and permanent cell cycle arrest. As a result of
their central role in cell cycle regulation, components of the RB pathway are targeted in
10
Figure 1.
GROWTH FACTORS:
STRESS:
p16 IN K 4 A
pl9ARF
p1
cyclin D/cdk4
mdm2
I
I
pRb
p5 3
I
E2F
p21CIP
S-phase
S-phase
Figure 1. The RB pathway and the p53 pathway both regulate S-phase. The RB pathway
regulates S-phase entry and progression in response to growth factor signaling. The p53
pathway is normally inactive, but can be activated to control S-phase in response to many
types of cellular stress. Both pathways are critical targets in the process of tumorigenesis.
Additionally, components of these two pathways can communicate with each other during
S-phase regulation.
the majority of cancers. Evidence of this is seen in the frequent inactivation of the tumor
suppressor protein pRB or the cdk inhibitor p16INK4A as well as the activation of cdk
activity through amplification of cdk4 or D-type cyclins.
THE E2F FAMILY OF TRANSCRIPTION FACTORS
Discovery of E2F and general properties
Studies involving mammalian DNA tumor viruses provided early information
regarding cell cycle progression, DNA replication, transcription and transformation.
Because of their relatively small genome sizes, these viruses often employ cellular factors
in processes such as viral genome replication and transcription, rather than encoding viral
factors to carry out these processes. By studying how viruses co-opt these processes, we
gained insight into how these cellular processes normally proceed and what cellular
proteins are involved. Examples of these viruses include SV40 polyomavirus, human
papilloma virus (HPV) and adenovirus.
Immediately after infection, adenovirus initiates a coordinated attack on the
cellular machinery to carry out viral transcription and replication, induction of cell cycle
progression and inhibition of apoptosis. The first viral transcript to be produced
following adenoviral infection is E1A, which in turn activates the expression of several
other early viral transcripts. To accomplish this, E1A recruits the efforts of a variety of
cellular proteins. In particular, expression of E1A stimulates transcription of the E2 gene
by recruiting a cellular activity to the promoter, termed the E2 promoter-binding factor
(E2F) (Kovesdi et al., 1986).
12
E2F is a family of transcription factors, which to date is encoded by eight genes.
E2F1-5 interact with the pocket protein family, and will be the primary focus of this
section (Figure 2) (Trimarchi and Lees, 2002). E2F6, E2F7 and E2F8 lack the domain
required for E2F to bind the pocket proteins and have been shown to repress transcription
independently (Cartwright et al., 1998; de Bruin et al., 2003; Di Stefano et al., 2003;
Logan et al., 2004; Maiti et al., 2005; Trimarchi et al., 1998). Several studies have
suggested that E2F6 as a member of the Polycomb complex (Attwooll et al., 2005;
Ogawa et al., 2002; Storre et al., 2002; Trimarchi et al., 2001). This complex was
originally characterized in Drosophila melanogaster, and regulates the transcription of
patterning genes such as the Hox genes (Ringrose and Paro, 2004). The mechanism by
which E2F7 and E2F8 repress transcription remains to be elucidated.
Concurrent with the identification of E2F, studies utilizing murine F9 embryonal
carcinoma (EC) cells identified a differentiation-regulated transcription factor (DRTF1)
that possesses a similar activity to E2F (La Thangue and Rigby, 1987; La Thangue et al.,
1990). This activity was substantially down-regulated when F9 EC cells were stimulated
to undergo differentiation. Further studies of DRTF1 led to the cloning of DRTF1polypeptide 1 (DPi) (Girling et al., 1993). There are two known mammalian DP genes,
DPi and DP2 (Ormondroyd et al., 1995; Rogers et al., 1996; Wu et al., 1995; Zhang and
Chellappan, 1995). Expression of DPi is generally greater than that of DP2. Although
no functional distinctions have been made regarding these two proteins, experiments
suggest that DP2 has a greater capacity to enter the nucleus than DPi (de la Luna et al.,
1996). E2F and DP genes are found in non-mammalian species where they also play key
roles in cell cycle progression and development (Ceol and Horvitz, 2001; Dimova et al.,
13
Figure 2.
ZI[
E2F1
E2F2
ZZI
[
·
·
~~
I
Il
E2F3A
i
II
E2F3B
II
11
E2F4
....... a·
[[
E2F5
El
= NLS
= DNA binding
I
II
I = dimerization
= transactivation and pocket protein binding
Figure 1. Six members of the E2F family of transcription factors interact with the tumor
suppressor protein pRb and/or the other pocket protein family members, p107 and p130.
I
2003; Frolov et al., 2001; Gutierrez et al., 2002; Page et al., 2001; Philpott and Friend,
1994).
E2F activity consists of a heterodimeric complex between one E2F subunit and
one DP subunit (with the exception of E2F7 and E2F8) (Helin et al., 1993b).
Dimerization of E2F and DP involves residues comprising a leucine zipper and a separate
domain known as the marked box. The marked box was originally identified as a highly
conserved region among E2Fs lying outside of the DNA binding domain (Lees et al.,
1993). This domain was first shown to interact with the adenoviral E4 ORF 6/7, which
facilitates the interaction between E2F and DP (Jost et al., 1996; Obert et al., 1994). To
date, there is no equivalent cellular factor.
The crystal structure of E2F/DP complex showed that it is a member of the
winged-helix family of transcription factors (Zheng et al., 1999). Although amino acid
conservation within the DNA binding domain as a whole is not high, the amino acids that
contact DNA bases are perfectly conserved among all E2Fs and DPs. Mapping and
mutagenesis studies identified a consensus DNA binding site of E2F in the E2 promoter
(Loeken and Brady, 1989; Murthy et al., 1985; Zajchowski et al., 1985). Further studies
showed that the binding site, TTTCGCGC, is also present in a number of cellular genes
(Hiebert et al., 1991; Mudryj et al., 1990).
Given the focus of early adenoviral studies, it is not surprising most classic E2F
target genes had roles in DNA replication and cell cycle progression. However, with the
sequencing of the genome and the development of techniques such as microarrays and
chromatin immunopercipitation (ChIP) the list of E2F targets has been greatly expanded
to include genes with functions in mitosis, apoptosis, DNA damage and differentiation
15
(Ishida et al., 2001; Kalma et al., 2001; Kel et al., 2001; Ma et al., 2002; Markey et al.,
2002; Muller et al., 2001; Polager et al., 2002; Ren et al., 2002; Stanelle et al., 2002;
Vernell et al., 2003; Wells et al., 2002). Consistent with these reports, mouse models
also support a role for E2F in differentiation and development (Cloud et al., 2002; Fajas
et al., 2002; Humbert et al., 2000a; Landsberg et al., 2003; Lindeman et al., 1998;
Rempel et al., 2000; Yamasaki et al., 1996). These experiments helped demonstrate that
E2F has critical roles in processes beyond basic cell cycle regulation.
At the carboxy-terminus of the E2F proteins are overlaying domains specifying a
transcriptional activation domain and pocket protein binding domain (Helin et al., 1992).
So intertwined are these two domains that the generation of mutations to separately
define them has proved difficult. E2Fs interact with multiple histone acetyltransferase
(HAT) complexes including GCN5 and Tip60 activate transcription of target genes (Lang
et al., 2001; Taubert et al., 2004). However, binding of the E2Fs by pocket proteins
masks the transcriptional activation domain and presumably precludes interactions
between the E2Fs and HATs. Pocket proteins also associate with histone
deacetyltransferases (HDACs) and histone methyltransferases to mediate transcriptional
repression through E2F (Brehm et al., 1998; Ferreira et al., 1998; Lai et al., 2001;
Macaluso et al., 2003; Magnaghi-Jaulin et al., 1998; Nicolas et al., 2000; Rayman et al.,
2002).
Although E2Fs collectively mediate both transcriptional activation and
transcriptional repression, these two functions are generally thought to be subdivided into
two classes of E2Fs (Trimarchi and Lees, 2002). E2F1, E2F2 and E2F3A are known as
the activating E2Fs because of their potent ability to induce transcription of E2F target
16
genes. E2F4 and E2F5 are known as the repressive E2Fs. The classification of these
E2Fs as either activators or repressors is based upon functional and structural
characterizations of the individual E2Fs. Additionally, there are key differences in the
subcellular localization, the expression patterns, and the regulation of the activating E2Fs
and the repressive E2Fs.
E2F1, E2F2, and E2F3A all possess over 100 amino acids amino terminal to their
DNA binding domain, whereas E2F3B, E2F4 and E2F5 possess only short stretches of
amino acids in this region. The significance of these amino-terminal sequences remains
unclear, as the sequence identity in this region is very poor. Closer to the DNA binding
domain lies a short sequence which confers cyclin A binding to E2F1-3 (Krek et al.,
1994; Xu et al., 1994). Although E2F4 and E2F5 do not share this direct interaction, they
indirectly associate with cyclin A through their interactions with p107 and p130 (Castano
et al., 1998; Chibazakura et al., 2004; Woo et al., 1997; Zhu et al., 1995).
Subcellular localization of E2Fs is controlled through cis-acting amino acid
sequences. E2F1-3 are constitutively nuclear due to the presence of a nuclear localization
sequence (NL,S) (Allen et al., 1997; Magae et al., 1996). It has not been demonstrated
whether certain circumstances exist during which they shuttle in and out of the nucleus.
In contrast, E2F4 and E2F5 are predominantly cytoplasmic (Verona et al., 1997). E2F4
possesses multiple nuclear export sequences (NES), characterized by many hydrophobic
leucine or isoleucine residues (Gaubatz et al., 2001). When in complex with pocket
proteins, the NES is masked, allowing for the presence of E2F4 and E2F5 in the nucleus.
When released by the pocket protein, E2F4 and E2F5 are rapidly exported out of the
nucleus. Interestingly, in cells lacking both p107 and p130, E2F4 and E2F5 are reported
17
to be unable to enter the nucleus (Rayman et al., 2002). These properties explain why
E2F4 and E2F5 are potent repressors but poor activators of transcription.
E2Fs are widely expressed, but the specific expression patterns of the individual
E2Fs in different tissue and cell types has been only partially characterized (Kusek et al.,
2000). The expression pattern of the E2Fs during the cell cycle has been more carefully
documented. E2FI, E2F2 and E2F3A are not expressed in quiescent cells. Transcription
of these genes initiates late in G1, and the promoters of these genes possess multiple E2F
and E-box sequences (bindings sites for the transcription factor c-myc), suggesting some
degree of positive feedback (Adams et al., 2000; Hsiao et al., 1994; Sears et al., 1997).
E2F4 and E2F5 are expressed throughout the cell cycle, with little fluctuation. Currently,
the transcription factors that regulate the promoters of these genes have not been
characterized.
E2F3B, which is discussed in more detail below, shows a pattern of
expression similar to E2F4 and E2F5 (Leone et al., 2000). Finally, recent reports indicate
that the E2Fs are translationally regulated by microRNAs, although this mechanism of
regulation is not well understood (O'Donnell et al., 2005). In addition to these types of
control, E2Fs are also regulated by multiple post-translational mechanisms including
ubiquitination, acetylation and phosphorylation (Campanero and Flemington, 1997;
Dynlacht et al., 1994; Dynlacht et al., 1997; Galbiati et al., 2005; Gaubatz et al., 2000;
Hallstrom and Nevins, 2003; Hateboer et al., 1996; Hofmann et al., 1996; Krek et al.,
1994; Martinez-Balbas et al., 2000; Marzio et al., 2000; Trouche et al., 1996; Xu et al.,
1994).
18
Models of E2F function
Many models have been proposed to explain the mechanism of E2F target gene
regulation, although there is not a consensus. One question addressed by various models
is whether E2F target gene expression is primarily regulated by activation or repression.
During cell cycle re-entry, there is a clear use of repression during quiescence and of
activation once cells re-enter the cell cycle. However, this may not model the regulation
of E2F target genes in cycling cells. In this setting, expression of E2F target genes may
be achieved by the binding of activating E2Fs to the promoters of target genes. However,
it is equally likely that expression of E2F target genes may be achieved by the removal of
repressive E2Fs from the promoters of target genes. There is data in support both
models, suggesting that the mechanism of target gene regulation may vary from gene to
gene.
Another question about E2F target gene regulation is the issue of regulation by
specific E2Fs versus overall E2F dosage. Some E2F target genes are believed to be
specifically regulated by a single E2F or perhaps a subset of E2Fs. In other cases, it
appears as if the determining factor is E2F dosage. The sum of activating E2Fs less the
sum of repressive E2Fs equals the E2F activity regulating a particular gene. Thus the
relative levels of' different E2Fs would determine E2F target gene expression. Again,
there is data consistent with both models. The question of compensation is also relevant
here. If there is normally specificity involved in the regulation of a given E2F target
gene, by one member of the E2F family, the absence of that E2F should be sufficient to
deregulate expression of the target gene. On the other hand, if the remaining E2Fs can
19
compensate for the loss of one E2F, this may obscure the correct understanding of target
gene regulation.
The E2F3 locus
My studies on E2F have focused primarily on one family member, E2F3. The
E2F3 locus specifies two gene products (Lees et al., 1993; Leone et al., 2000). The two
transcripts encoded by this locus utilize distinct first exons, but share the remaining exons
and are read in the same reading frame. Consequently, the two E2F3 proteins are
identical except at their amino terminus. One hundred twenty-two amino acids at the
amino terminus of E2F3A are replaced by six novel amino acids in E2F3B. However, all
known domains in E2F3A are present in both proteins and no function has yet been
assigned to the amino terminus of E2F3A.
E2F3A and E2F3B show different patterns of expression (Leone et al., 2000).
E2F3A is not expressed in quiescent cells. As cells proceed from G1 into S-phase,
expression of E2F3A is induced. The promoter of E2F3A contains several E2F
consensus binding sites as well as E-boxes (Myc binding sites) (Adams et al., 2000). In
contrast, E2F3B is expressed in both quiescent and cycling cells, and its levels do not
vary during the cell cycle. The promoter of E2F3B contains several Ets and Spl
consensus binding sites.
E2F3A has been extensively characterized as a transcriptional activator and a
positive regulator of cell cycle progression (Lees et al., 1993). Once quiescent cells reenter the cell cycle, E2F3 binding activity accumulates in late GI and then decreases as
cells exit S-phase (Leone et al., 1998). However, E2F3 binding activity reappears at the
20
following G /S transition, indicating that E2F3A plays a recurring role during each cell
cycle. Additionally, overexpression of E2F3A induces S-phase entry and causes
quiescent cells to re-enter the cell cycle (Lees et al., 1993).
There is considerable disagreement on whether expression of E2F3A can also
induce apoptosis, and the results appear to be highly influenced by the method of
overexpression that is used. Some studies suggest that the ability to induce apoptosis is
an E2F1-specific function not shared by E2F3, while others suggest that E2F3 can also
induce this pathway (DeGregori et al., 1997; Leone et al., 2001). Overexpression of
E2F3A in the pituitary induces abnormal proliferation of previously quiescent
melanotroph cells (Denchi et al., 2005). However, these cells cells do not undergo
apoptosis, but instead ultimately cease cell division and enter a senescent-like state. This
suggests that cells are actively monitoring E2F activity, and in some tissues additional
mutations would be necessary for de-regulated E2F activity to promote tumorigenesis.
Mouse embryonic fibroblasts (MEFs) lacking both isoforms of E2f3 display
impaired cellular proliferation and this phenotype is exacerbated when cells are grown at
lower densities (Humbert et al., 2000b). Additionally, E2f3-deficient MEFs driven into
quiescence by serum deprivation re-enter the cell cycle following the re-addition of
serum with decreased efficiency. This is apparently due to a delay in S-phase entry as
well as a defect in S-phase progression. Decreased/delayed expression of E2F target
genes correlates with this observation. MEFs lacking E2fl-3 exhibits more profound
defects in proliferation and cell cycle re-entry, suggesting that E2F1 and E2F2 can also
influence these phenotypes but to a lesser degree than E2F3 (Wu et al., 2001a).
21
Protein-protein interactions may explain some reason for the specific importance
of E2F3 in the regulation of cellular proliferation.
A yeast two hybrid using the marked
box of various E2Fs identified RYBP as a specific interactor of E2F2 and E2F3 (Schlisio
et al., 2002). This interaction positively regulates the Cdc6 promoter. A similar
approach was used to identify TFE3 as a binding partner of E2F3 (Giangrande et al.,
2003; Giangrande et al., 2004). Together, E2F3 and TFE3 coordinate the expression of
several genes, including the p68 subunit of DNA polymerase alpha and ribonucleotide
reductase. Regulation of these genes appears to be cooperative, as MEFs lacking either
E2f3 or Tfe3 show similar defects in gene regulation. However, not all genes affected by
the loss of E2f3 were also affected by the loss of Tfe3, and generally the loss of E2f3
resulted in a more severe defect.
Given the key role of E2F3 as a regulator of cellular proliferation and the highly
proliferative state of tumors, it is not surprising to know that E2F3 protein is abundant in
many types of cancer. However, more recent evidence suggests that E2F3 may be
specifically targeted in some tumors. Bladder cancer often displays amplification of
chromosome 6p22, which contains the E2f3 locus (Veltman et al., 2003). In both bladder
and prostate tumors, high E2F3 expression correlates with poor prognosis and invasive
cancer (Feber et al., 2004; Foster et al., 2004; Oeggerli et al., 2004; Veltman et al., 2003).
Metagene analysis has been used to model the downstream activity of E2F3 relative to
other E2Fs, and may provide some clues to its specific role in tumorigenesis (Huang et
al., 2003).
Mouse models have been used to further probe the function of E2F3. Germline
deletion of E2f3 in mice results in complete embryonic lethality in pure strain
22
backgrounds, and partial lethality in mixed strain backgrounds (Cloud et al., 2002;
Humbert et al., 2000b). Analysis of pure strain background E2f3-deficient embryos
reveals cardiac defects. Additionally, E2fl;E2f3 compound mutant mice display earlier
embryonic lethality, as early as e9.5 (Cloud et al., 2002). This suggests that the
transcriptional activation is critical for proper embryo development. Surviving adult E2f3
knockout mice in the mixed strain backgrounds eventually succumb to dilated
cardiomyopathy (DCM), although the cause remains unknown. Unlike E2fl-deficient
mice, E2f3 knockout mice are not prone to spontaneous tumor development (Yamasaki et
al., 1996). Nevertheless, the role of E2F3 in cellular proliferation suggests that it could
play a positive role in tumorigenesis as a result of deregulation of the RB pathway.
REGULATION OF E2F BY THE RB PATHWAY
Rb-i was first identified as an important tumor suppressor gene mutated in cancer
(Friend et al., 1986; Fung et al., 1987; Lee et al., 1987). Humans with a germline
mutation in the Rb-1 gene develop spontaneous bilateral retinoblastoma. These
individuals are also prone to secondary tumors such as osteosarcoma (Fletcher et al.,
2004). Additionally, pRB is functionally inactivated in a majority of other tumor types.
Concurrent with studies of retinoblastoma, pRB was also identified as a key interacting
protein of viral oncoproteins such as adenovirus E1A, human papilloma virus (HPV) E6
and SV40 large T antigen (Dyson et al., 1992; Dyson and Harlow, 1992). These viral
gene products bind and inactivate pRB and its pocket protein family members (Ewen et
al., 1991; Li et al., 1993; Mayol et al., 1993). The importance of pRB in tumor biology is
23
illustrated by fact that it is both a target of mutation as well as inactivation by
transforming viral oncoproteins.
General properties of the pocket protein family
pRB, p107 and p130 comprise the pocket protein family, so named for a
conserved region through which many of their known interactions and functions occur.
The small pocket consists of an A domain and a B domain separated by a spacer region.
It binds to an LxCxE motif found in many known pocket protein interactors. The crystal
structure of this interaction has been solved (Lee et al., 1998). The E2F transcription
factor does not possess an LxCxE motif, and interacts with a large pocket consisting of an
additional C domain (Helin et al., 1992; Shan et al., 1996).
Endogenous E2Fs and pocket proteins exhibit specificity among their preferred
protein-protein interactions. pRB interacts with E2F1, E2F2, E2F3A and E2F3B; p107
interacts primarily with E2F4; and p130 interacts with both E2F4 and E2F5 (Moberg et
al., 1996). The reason for this specificity is not known, nor is whether it contributes to
pRB's role as a tumor suppressor protein. However, re-organization of pocket protein
complexes has been observed in cells lacking pRB (Callaghan et al., 1999; Lee et al.,
2002). This is seen in MEFs, differentiated neuronal stem cells, and in the pituitary. In
these settings, E2F1 and E2F3 associate with p130 and p107 respectively.
This
phenomenon is exacerbated in E2f4;Rb double knockout cells and may be critical for the
ability of these cells to effectively exit the cell cycle.
24
Regulation of the pocket protein family
The expression pattern of the three pocket proteins is differentially regulated
(Classon and Harlow, 2002). The expression of pRB does not vary significantly during
the cell cycle or upon cell cycle exit. p107 is not expressed in quiescent cells, but its
expression is induced during the G1/S transition as a result of transcriptional regulation
by E2F. In contrast, p130 is highly expressed in quiescent and differentiated cells. Upon
cell cycle re-entry, p130 levels decrease as a result of ubiquitin-mediated degradation by
the proteasome (Bhattacharya et al., 2003).
As mentioned earlier, regulation of the E2F and pRB interaction occurs primarily
via pocket protein phosphorylation (Classon and Harlow, 2002). This change in
phosphorylation status affects the ability of pRB to associate with many of its protein
interactors. In GO or Gi cells, pRB is present in a hypo-phosphorylated
state. Sequential
phosphorylation events by cyclin-cdk complexes during cell cycle progression lead to the
accumulation of' hyper-phosphorylated
pRB. Mutation of cdk phosphorylation sites on
pRB results in a constitutively active pRB (Knudsen and Wang, 1997). Both p107 and
p130 are also substrates for cyclin-cdk phosphorylation.
Regulation of E2F by the pocket protein family
Pocket proteins inhibit E2F target gene expression through at least two
mechanisms. First, the pocket protein binding domain of E2F overlaps with its
transactivation domain leading to passive repression of E2F when the two proteins
interact (Helin et al., 1993a). Second, pocket proteins interact with several different
histone and chromatin modifying enzymes to actively regulate gene expression. The
pocket proteins interact with histone deacetylases (HDACs) as well as histone
25
methyltransferases such as Suv39H1 and Suv39H2 (Brehm et al., 1998; Ferreira et al.,
1998; Lai et al., 2001; Macaluso et al., 2003; Magnaghi-Jaulin et al., 1998; Nicolas et al.,
2000; Rayman et al., 2002). Pocket proteins also interact with nucleosome remodeling
proteins such as Brgl and Brm (Strobeck et al., 2000; Strobeck et al., 2002). The
expression of these histone and chromatin modifying enzymes has been shown to be
essential for pRB to block cell cycle progression.
There appear to be differences in the repression of E2F target genes mediated by
pRB and that mediated by p107 and p130. Chromatin immunopercipitation
(ChIP)
experiments suggest that p107 and p130 bind the promoters of E2F target genes during
the cell cycle (Rayman et al., 2002; Takahashi et al., 2000). In contrast, pRB cannot be
found to be associated with the promoters of E2F target genes during the cell cycle.
Instead, pRB looks to be involved in permanent repression of gene expression.
pRB co-
localizes with senescence-associated heterochromatic foci (SAHFs) (Young and
Longmore, 2004). Additionally, pRB positively regulates the activity of several
differentiation specific transcription factors and can be found at the promoters of target
genes involved in differentiation.
In addition to playing a key role in events during the cell cycle, the pocket
proteins and E2F have an important function in the decision to exit the cell cycle.
Deletion of both Rb and p107 results in bypass of the serum-dependent restriction point
(Gad et al., 20041). Interestingly, acute ablation of Rb but not the germline deletion
renders cells incompetent to exit the cell cycle in response to serum deprivation (Sage et
al., 2003). The may be explained by the up-regulation of p107 observed in the germline
deletion of Rb, and the ability of p107 to compensate for pRB in this setting. Cells
26
deficient for all three pocket proteins fail to arrest in response to serum deprivation,
contact inhibition or the loss of adhesion and are immortal (Dannenberg et al., 2000; Sage
et al., 2000). Reintroduction of pRB into cell lines lacking Rb-1 induces a senescence-
like state (Alexander and Hinds, 2001; Tiemann and Hinds, 1998; Xu et al., 1997).
Additionally, the acute loss of Rb in senescent cells is sufficient to allow them to re-enter
the cell cycle (Sage et al., 2003).
Pocket protein family mouse models
Mice heterozygous for the Rb gene do not develop retinoblastoma like their
human counterparts. Instead, they succumb to spontaneous tumors of the pituitary and
thyroid (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). Rb chimeric animals
exhibit the same tumor predisposition (Williams et al., 1994b). Animals lacking p107 or
p130 are not prone to spontaneous tumor development. However, ablation of Rb in the
background of pl07 or p130-deficiency
gives rise to retinoblastoma (Chen et al., 2004;
MacPherson et al., 2004b). This suggests that p107 and p130 are playing "back-up" roles
as tumor suppressors. Viral oncogenes have also been used to functionally inactivate the
pocket proteins. For example, the T121 mouse expresses a transgene containing a
truncated version of the SV40 large T antigen. This mouse has been used to characterize
the role of the pocket proteins in tumor development in several different tissues (Pan et
al., 1998; Simin et al., 2004; Xiao et al., 2002).
Knockout mouse strains have been used to determine the contribution of specific
E2Fs in various tumor models. Rb heterozygous mice that are also deficient for E2fl
possess a greatly extended the lifespan, supporting the conclusion that E2F is deregulated
in Rb-deficient tumors (Yamasaki et al., 1998). This appears to be a general property of
27
the activating E2Fs, as the deletion of E2f3 similarly improved the survival of Rb
heterozygous mice (Ziebold et al., 2003). Interestingly, the loss of E2f3 also enhanced
the aggressiveness of thyroid tumors in these mice, demonstrating that E2Fs can have
both tumor promoting and tumor restricting functions. Finally, the loss of E2f4 also
inhibits tumor development in Rb-heterozygous mice (Lee et al., 2002). This has been
attributed to the redistribution of E2Fs and pocket proteins, leading to inhibition of E2F1
and E2F3 by p130 and p107, respectively.
Unfortunately, this does not tell us whether
E2F4 has a direct function in these tumors.
Examination of Rb-deficient cells and embryos has provided additional evidence
that deregulation of E2F activity is a key consequence of Rb-loss. Germline deletion of
Rb leads to inappropriate S-phase entry in the central nervous system (CNS) and
increased E2F activity (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). The
additional deletion of either E2fl or E2f3 reduces inappropriate S-phase entry (Tsai et al.,
1998; Ziebold et al., 2001). Similarly, MEFs lacking Rb have a shortened G1 and
elevated levels of specific E2F target genes including p107 and cyclin El (Hurford et al.,
1997). Recently, Rb-deficient MEFs have been shown to possess a defect in mitotic
progression due to deregulation of Mad2 (Hernando et al., 2004). MEF lacking both
p107 and p130 also show defects in E2F target gene regulation that are distinct from
those regulated by pRB (Hurford et al., 1997). Also, plO07-deficient MEFs display
elevated Skp2 expression (Rodier et al., 2005). These experiments demonstrate that E2F
is a key downstream component of the RB pathway.
28
Cyclins and cdks
Sequential phosphorylation events during Gi and the transition into S-phase
regulate pRB function. The cyclins and cyclin dependent kinases are responsible for
phosphorylation of pRB, p107 and p130 are described below.
D-type cyclins (D1, D2, and D3 in mammals) function in early G1 (Sherr, 1993).
In addition to their role in cell cycle progression, D-type cyclins may also promote
cellular growth (Datar et al., 2000; Meyer et al., 2000; Meyer et al., 2002). Kinase
activity requires complex assembly between D-type cyclins and either cdk4 or cdk6
(Matsushime et al., 1992; Meyerson and Harlow, 1994). The critical target of these
kinases is pRB, and few other targets have been identified. The expression of D-type
cyclins in response to growth factor stimulation occurs through the Ras pathway (Gille
and Downward, 1999). Overexpression of D-type cyclins causes a shortening in the
length of G1 (Quelle et al., 1993). Recently, MEFs have been generated which lack all
three D-type cyclins (Kozar et al., 2004). Proliferation of the D-type cyclin triple
knockout (TKO) MEFs is only slightly impaired and the expression of other cell cycle
regulators appears to be unaffected. Although the overall phosphorylation of pRB is
reduced in the D-type cyclin TKO MEFs, two presumed D-type cyclin specific sites
remain efficiently phosphorylated. Cdk4;cdk6 DKO MEFs have also been generated,
and they have similar properties to those observed in the D-type cyclin TKO MEFs
(Malumbres et al., 2004).
E-type cyclins (El and E2 in mammals) function in late Gi and during S-phase
(Sherr, 1993). Kinase activity requires complex assembly between E-type cyclins and
cdk2. In addition to further phosphorylating pRB, E-type cyclins possess other key
29
targets with roles in cell cycle progression such as p27KIP1 (Sheaff et al., 1997). E-type
cyclins also regulate centrosome duplication through the phosphorylation of
nucleophosmin (Okuda et al., 2000). The expression of cyclin E is normally under the
control of the E2F transcription factor. Overexpression of E-type cyclins accelerates
progression from Gi into S-phase and can substitute for the activity of cyclin D (Geng et
al., 1999; Ohtsubo and Roberts, 1993). Recently, MEFs have been generated that lack
both E-type cyclins (Geng et al., 2003; Parisi et al., 2003). Although E-type cyclin DKO
MEFs display a subtle proliferation impairment, expression of other components of the
cell cycle machinery as well as overall phosphorylation levels of pRB remain unaffected.
There are also no centrosomal abnormalities observable in E-type cyclin DKO MEFs.
Cdk2 knockout MEFs have also been generated, and have similar properties to those
observed in the E-type cyclin DKO MEFs (Berthet et al., 2003; Ortega et al., 2003).
Cdk inhibitory proteins
Two families of cyclin dependent kinase inhibitor proteins regulate cyclin
dependent kinase activity. They cooperate with the pocket proteins to mediate repression
of E2F and cell cycle arrest.
The INK4 family is comprised of p16INK4A, p15INK4B, p18INK4C and
pl9INK4D (Ortega et al., 2002). These proteins specifically associate with cdk4 and
cdk6, and prevent the association of cdk4 and cdk6 with the D-type cyclins. Members of
the INK4 family are capable of inducing G1 arrest when over-expressed, but this activity
is dependent on functional pRB. The regulation of the INK4 family members is still
being characterized, but it is clear that they possess distinct patterns of expression during
development and respond differently to various growth inhibitory signals. For example,
30
p16INK4A expression becomes increasingly elevated as cells accumulate population
doublings and undergo senescence. Premature senescence induced by activation of the
Ras pathway also leads to expression of p16INK4A, mediated by the Ets transcription
factor (Ohtani et al., 2001).
The CIP/KIP family is comprised of p21CIP, p27KIP1 and p57KIP2 (Sherr and
Roberts, 1999). These proteins inhibit cdk2 kinase activity, but do so without
disassociating cdk2 from E-type and A-type cyclins. Instead, they form a tri-molecular
complex. The CIP/KIP family is believed to perform an additional function, promoting
the assembly of D-type cyclins with cdk4 or cdk6 (Cheng et al., 1999). The generation of
p21CIP;p27KIP1 double knockout MEFs suggests that this may not be an essential
function for the CIP/KIP family (Bagui et al., 2003; Sugimoto et al., 2002). Additionally,
it was thought that D-type cyclins sequestered members of the CIP/KIP away from Etype and A-type cyclins. However, D-type cyclin TKO MEFs do not possess any
impairment in cdk2 activity(Kozar et al., 2004). Like the INK4 family, there is a distinct
pattern of expression of CIP/KIP family members in response to various growth
inhibitory signals. For example, p21CIP is a major downstream transcriptional target of
the p53 tumor suppressor protein, serving as a critical link between these two pathways
(Bunz et al., 1998; el-Deiry et al., 1993; Niculescu et al., 1998).
THE p53 PATHWAY
Discovery of p53 and general properties
p53 was originally identified by several studies as a cellular protein that bound to
the large T antigen in SV40 polyomavirus infected cells (Chang et al., 1979; Kress et al.,
31
1979; Lane and Crawford, 1979; Linzer and Levine, 1979; Linzer et al., 1979).
Subsequent work demonstrated that p53 was also associated with El B of adenovirus and
E6 of human papillomavirus (HPV) (Sarnow et al., 1982; Scheffner et al., 1990). It was
also noted that p53 levels, though variable, were often more abundant in transformed
cells, whereas non-transformed cells possess very small amounts of this protein (Mowat
et al., 1985). Due to the fact that p53 was initially cloned from cells possessing a
dominant negative missense mutation, p53 was first thought to function as an oncogene
(Jenkins et al., 1985). When later studies uncovered this fact, the wild-type protein was
quickly established as a potent tumor suppressor. Mutation of p53 was linked to a
hereditary cancer, Li-Fraumeni syndrome, and a plethora of spontaneous p53 mutations
have been identified in other cancers (Malkin et al., 1990; Olivier et al., 2004; Pfeifer,
2000).
The p53 tumor suppressor protein is a strong activator of transcription.
The
amino-terminus of the p53 protein contains two domains, a transactivation domain and a
proline-rich domain. The transactivation domain is highly acidic and of comparable
strength to other strong activation domains (Fields and Jang, 1990; Unger et al., 1993).
The proline-rich domain consists of five PXXP motifs. This domain is similar to motifs
found in SH3-binding proteins. The role of the proline-rich domain has not been
convincingly established (Edwards et al., 2003; Zhu et al., 1999; Zhu et al., 2000).
The central region of p53 contains the DNA binding domain. This is the site of
an overwhelming majority of p53 mutations (Olivier et al., 2004; Pfeifer, 2000). The Xray crystal structure of the DNA binding domain bound to DNA has been solved,
confirming the importance of many of the mutations observed in cancers (Cho et al.,
32
1994; Zhao et al., 2001). The consensus p53 binding site consists of two copies of the
sequence 5'-Pu-Pu-Pur-C-(A/T)-(T/A)-G-Py-Py-Py-3'
that are each separated by 0-13
nucleotides (el-Deiry et al., 1992). They are often located in the 5' untranslated region as
well as the first two introns of target genes.
The carboxy-terminus of p53 contains the oligomerization domain and a proposed
regulatory region that is highly basic. The p53 protein forms tetramers and
oligomerization is required for DNA binding and transcriptional regulation of target
genes, although DNA is not required for tetramer formation (Friedman et al., 1993;
Kraiss et al., 1988). The X-ray crystal structure of the oligomerization domain has been
solved (Jeffrey et al., 1995). The carboxy-terminus of p53 also contains a nuclear export
sequence (NES) and three nuclear localization signals (NLS). Tetramerization is reported
to mask the NES, encouraging nuclear localization of the protein (Stommel et al., 1999).
Although p53 is best characterized as a transcriptional regulator, some recent studies
have proposed an additional cytoplasmic role for p53 (Chipuk et al., 2004; Marchenko et
al., 2000; Mihara et al., 2003; Sansome et al., 2001; Zhao et al., 2005). The relevance of
this activity remains to be proven.
There are two additional p53 family members, p63 and p73, which bear overall
domain similarity to p53 (Melino et al., 2003; Moll and Slade, 2004). However, p63 and
p73 share more homology with each other than they do to p53. Additionally, p63 and
p73 are highly alternatively spliced. It is not yet clear what functions the differenet
isoforms possess. However, those lacking the amino-terminal activation domain may
function as natural dominant negative proteins. p53 was not thought to share this
property, but recently it has been demonstrated that p53 is also alternatively spliced
33
(Courtois et al., 2004). Although evidence of p63 and p73 mutation in cancer is rare,
especially compared to that of p53, analysis of p63,p73 compound mutant mice suggest
that these two genes do possess tumor suppressive function (Flores et al., 2005). The
nature of this function remains largely unclear as these proteins likely both share a
common subset of target genes with p53 as well as possess their own unique target genes.
This hypothesis was recently supported by studies of p53;p63,p73 compound mutant
cells (Flores et al., 2002). p 6 3 and p73 function may also be altered in the presence of
mutant p53 (Di Como et al., 1999; Gaiddon et al., 2001; Marin et al., 2000; Olive et al.,
2004; Strano et al., 2000).
p53 function and target genes
Expression of p53 results in cell cycle arrest or the induction of apoptosis. p53
controls the expression of target genes that function in both these processes. To date, it is
not clear how the decision is made to promote cell cycle arrest over apoptosis or vice
versa. Little information on this question has been gained through the analysis of p53
DNA binding sites, suggesting that the answer lies either in specific post-translational
modifications, p53 protein-protein interactions, or the coordinated activation of parallel
pathways. Regardless, characterization of p53 target genes has led to a better
understanding of the part played by p53.
Expression of p53 leads to cell cycle arrest in both G1 and G2 (Iliakis et al., 2003;
Kuerbitz et al., 1992; Taylor and Stark, 2001). A number of p53 target genes have been
implicated in the mediation of cell cycle arrest. The cyclin dependent kinase inhibitor
p21CIP has been the most well-characterized (Bunz et al., 1998; el-Deiry et al., 1993;
Niculescu et al., 1998). In addition to its ability to inihibit cyclin-dependent kinase
34
activity, p21CIP also interacts with PCNA. Because PCNA plays a key role in DNA
replication, it is believed that this also represents an important contribution of p21CIP
during cell cycle arrest. MEFs deficient forp2lCip
fail to undergo p53-dependent cell
cycle arrest (Brugarolas et al., 1995; Deng et al., 1995). These cells also proliferate more
rapidly than wild-type cells, but unlike p53 knockout MEFs, p21Cip knockout MEFs still
undergo senescence. GADD45 is another p53 target gene involved in mediating cell
cycle arrest. (Papathanasiou et al., 1991) Expression of GADD45 prevents the transition
from G2 into mitosis by inhibiting cdc2 kinase activity. Another target of p53 is 14-3-3
sigma, which is also involved in regulation of the G2/M transition following cellular
stress (Hermeking et al., 1997).
Induction of apoptosis by p53 involves target genes that function at the plasma
membrane as well as in the mitochondria (Fridman and Lowe, 2003). DR5, PERP and
Fas-ligand are p53 target genes involved in the extracellular signaling pathways that
induce apoptosis (Attardi et al., 2000; Owen-Schaub et al., 1995; Wu et al., 1997). Bax,
Puma, Noxa, and Bid are involved in apoptosis conducted through the mitochondria
(McCurrach et al., 1997; Nakano and Vousden, 2001; Oda et al., 2000; Sax et al., 2002;
Yin et al., 1997; Yu et al., 2003). The single deletion of Bax, Puma, or Bid results in
partial resistance to p53-dependent apoptosis, although clearly multiple target genes are
able to effect p53-dependent cell death. Apaf- 1 is a p53 target gene that functions
through the regulation of caspase activation (Moroni et al., 2001; Soengas et al., 1999).
Deficiency in Apaf-l or caspase 9 also impairs p53-dependent apoptosis.
35
Regulation of p53 by post-translational modification
Since p53 activation can have serious consequences for cell cycle progression and
cell viability, it is critical that p53 is properly regulated. Control of p53 activity occurs
primarily through the post-translational modification of the p53 protein (Appella and
Anderson, 2001; Bode and Dong, 2004). Levels of p53 are low in unstressed, nontransformed cells. However, p53 is rapidly stabilized following cellular stress including
but not limited to ultraviolet (UV) irradiation, gamma-irradiation, nucleotide depletion,
hypoxia, hyperoxia, heat shock and senescence. Post-translational modifications that
have been identified on p53 include acetylation, glycosylation, methylation, neddylation,
phosphorylation, ribosylation, sumoylation, and ubiquitination. These modifications are
typically associated with changes in p53 protein stability and p53 DNA binding activity.
It is not clear whether these modifications are also responsible for influencing p53
transcriptional activity and/or target gene selection. The role of phosphorylation and
ubiquitination in p53 regulation has received the most attention and is consequently the
most well-understood.
Ubiquitination of p53 is responsible for keeping steady-state p53 protein levels
low, thereby also keeping p53 activity low. Murine double minute 2 (Mdm2) is an E3
ubiquitin ligase that binds to the amino-terminus of p53 (Haupt et al., 1997; Honda et al.,
1997; Kubbutat et al., 1998; Kubbutat et al., 1999; Momand et al., 1992; Oliner et al.,
1993). The interaction of mdm2 with p53 inhibits the function of the p53 transactivation
domain and decreases the stability of p53. Mdm2 is amplified in many cancers (Oliner et
al., 1992). Mdm2 is a p53 target gene, and this permits p53 to regulate its own protein
levels (Wu et al., 1993). Mdm2 can also auto-ubiquitinate (Honda and Yasuda, 1999).
36
Mdm2 additionally interacts with E2F and pRB (Hsieh et al., 1999; Loughran and La
Thangue, 2000; Sdek et al., 2004; Wunderlich et al., 2004). The importance of the
Mdm2-p53 regulatory loops is underscored by the early embryonic lethality of Mdm2deficient embryos. This lethality is completely rescued in Mdm2;p53 double knockout
embryos, demonstrating the essential role of Mdm2 in the regulation of p53 protein levels
and p53 activity (Jones et al., 1995; Montes de Oca Luna et al., 1995). Finally, the
Mdm2 homolog MdmX also regulates p53, although it does not possess intrinsic E3
ubiquitin ligase activity (Marine and Jochemsen, 2005).
HPV E6 targets p53 for ubiquitin-mediated degradation by interacting with the
cellular E6 associated protein (E6AP) (Huibregtse et al., 1991; Huibregtse et al., 1993).
E6AP is an E3 ubiquitin ligase (Scheffner et al., 1993). Interestingly, E6AP only
promotes the ubiquitin-mediated degradation of p53 in HPV infected cells (Talis et al.,
1998). It appears that E6AP is not normally involved in p53 regulation.
Phosphorylation of p53 is responsible for switching on p53 activity, by increasing
protein stability and/or DNA binding activity. DNA damage leads to the activation of the
kinase ataxia telangiectasia mutated (ATM) and AT-related (ATR). Atm is mutated in the
disease ataxia telangiectasia (A-T). Individuals with this disease develop a range of
maladies, including cancer. ATM and ATR directly phosphorylate p53 on residue serine
15 in humans and the equivalent residue (serine 18) in mice (Banin et al., 1998; Canman
et al., 1998; Saito et al., 2002; Siliciano et al., 1997; Tibbetts et al., 1999). Cells from AT patients have an impaired DNA damage response and do not stabilize p53
appropriately, consistent with the role of this kinase, and this phenotype is mirrored in
Atm-deficient mice (Barlow et al., 1996). Other kinases have also been implicated in the
37
phosphorylation of this residue, including p38, Erk, and DNA-PK (She et al., 2000; Shieh
et al., 1997). Knock-in mice have been generated using the endogenous p53 locus in
which serine 18 has been substituted with an alanine (Chao et al., 2003; Sluss et al.,
2004). This mutation resulted in decreased p53 target gene activation following DNA
damage, although no change in p53 protein stabilization or DNA binding was reported.
They also showed a reduced apoptotic response in thymocytes isolated from these mice,
although there was no evidence of spontaneous tumor development. Additionally, this
mutation appeared to have no effect on proliferation or the ability of these cells to
undergo G1 arrest.
Chkl and Chk2 are kinases that are activated down-stream of ATM and ATR and
independently phosphorylate p53 on serine 20 in humans and the equivalent residue
(serine 23) in mice (Bell et al., 1999; Chehab et al., 2000; Chehab et al., 1999; Shieh et
al., 1999; Unger et al., 1999; Wu et al., 2001b). The importance of these kinases is
supported by evidence showing that a small fraction of Li-Fraumeni patients harbor
mutations in Chk2 rather than p53. Additionally, Chk2-deficient thymocytes are
impaired in their ability to undergo apoptosis following DNA damage (Hirao et al.,
2000). Chk2 knockout cells do not appropriately stabilize p53 or induce p53 target gene
expression following DNA damage. MAPKAP2 has also been implicated in
phosphorylation of this residue (She et al., 2002). Knock-in mice have been generated
using the endogenous p53 locus in which serine 23 has been substituted with an alanine
(MacPherson et al., 2004a; Wu et al., 2002). MEFs derived from these mice displayed
normal p53 stabilization, activation of p53 target gene expression, and cell cycle arrest
following DNA damage. However, decreased p53 stabilization and apoptosis resistance
38
was observed in thymocytes and the cerebellum. Additionally, these mice eventually
developed lymphomas of B-cell origin.
To date there at least 17 serine and threonine residues on p53 reportedly
phosphorylated by 18 different kinases (Bode and Dong, 2004). It is difficult to know the
importance of each of these sites, or even of each kinase, and for most, the biological
relevance has yet to be established. One other mouse model involves a knock-in at the
serine 389 residue in the endogenous p53 locus (Bruins et al., 2004). This residue has
been shown to become phosphorylated specifically in response to UV-irradation but not
gamma-irradiation (Kapoor and Lozano, 1998; Lu et al., 1998). Cells from these mice
appear to be generally defective in responding to DNA damage involving nucleotide
excision repair (NER) (Hoogervorst et al., 2005). A kinase has not been definitely
identified as being responsible for phosphorylation on this site. In the future, remaining
phosphorylation sites will need to be confirmed, as will other p53 protein modifications.
Regulation of p53 by p19ARF
The tumor suppressor protein p19ARF, which regulates p53 as part of the tumor
surveillance response, is encoded by the INK4A locus. The INK4A locus was originally
identified as the gene encoding the p16INK4A tumor suppressor protein, a part of the RB
pathway. Subsequently, it was discovered that this locus encodes a second protein, the
alternate reading frame protein (ARF) (Quelle et al., 1995). In humans it is known as
pl4ARF; in mice it is known as pl9ARF.
The ARF protein utilizes a distinct first exon
from that used to encode p16INK4A. The resulting protein is read as a separate reading
frame. So although pl6INK4A and p19ARF share genomic DNA sequence, they bear no
amino acid similarity.
39
Expression of p19ARF was shown to arrest cells in both Gi and G2 phases of the
cell cycle (Quelle et al., 1995). The function of p19ARF was demonstrated to primarily
involve regulation of p53. This occurs through its interaction with mdm2 (Honda and
Yasuda, 1999; Kamijo et al., 1998; Kurokawa et al., 1999; Pomerantz et al., 1998; Tao
and Levine, 1999; Zhang et al., 1998). Most studies support the idea that p19ARF
inhibits mdm2 by sequestering it in the nucleolus (Weber et al., 1999). However, recent
studies have also established that p19ARF has additional roles beyond the regulation of
mdm2 (Kuo et al., 2003; Weber et al., 2000). Many of these alternate functions are still
consistent with its function in the nucleolus, as well as its role in mediating cell cycle
arrest and apoptosis. p19ARF has been shown to influence rRNA processing (Sugimoto
et al., 2003). pl9ARF also associates with nucleophosmin/B23 (Bertwistle et al., 2004;
Korgaonkar et al., 2005). One recent study suggests that p19ARF can regulate apoptosis
independently of p53 through a mechanism involving Bax and the mitochondria
(Nakazawa et al., 2003; Suzuki et al., 2003). p19ARF has been shown to regulate E2Fs
by targeting them for degradation(Martelli et al., 2001). p19ARF can also influence RhoGTPases (Guo et al., 2003; Guo and Zheng, 2004). Finally, p19ARF negatively regulates
the transcription factor FoxM1B and this interaction may be critical in hepatocellular
carcinoma development (Kalinichenko et al., 2004).
Several proteins have been implicated in the regulation of p19ARF, although
there is still much remaining to be clarified. p19ARF transcription can be induced by a
number of proteins, including several oncogenes, such as E1A, c-myc, ras, E2F, AP-1,
Pokemon, TBX2, TBX3 and Bmi-1 (Ameyar-Zazoua et al., 2005; Brummelkamp et al.,
2002; de Stanchina et al., 1998; Dimri et al., 2000; Jacobs et al., 2000; Jacobs et al.,
40
1999; Lingbeek et al., 2002; Molofsky et al., 2005; Palmero et al., 1998; Park et al., 2003;
Zindy et al., 1998). Additionally, p19ARF expression has been shown to be stimulated
by ATM (Li et al., 2004). More recently, CARF was identified as a p19ARF interacting
protein that collaborates with p19ARF through an unknown mechanism (Hasan et al.,
2002). p19ARF has also been shown to be itself regulated by ubiquitination (Kuo et al.,
2004).
Regulation of the p53 pathway by E2F
Several studies have implicated E2F in the regulation of pl9ARF. E2F consensus
binding sites can be found within the promoter of both pl9Arf and pl4Arf (Bates et al.,
1998). Conservation of these sites suggests that they are functionally relevant. Despite
this fact, mutation of these sites rendered no phenotype in reporter assays; the pl9Arf
promoter was E2F-responsive even when these sites were eliminated. It was further
demonstrated that E2F1 and E2F2 are not required for the induction of p19ARF
expression (Palmero et al., 2002). Another study showed that E2F may normally be
involved in repression of p19ARF expression (Rowland et al., 2002). Finally, two
separate mouse models in which both E2fl and p53 play critical roles were used to
illustrate that p19Arf does not form an obligate link between these genes (Tolbert et al.,
2002; Tsai et al., 2002a).
E2F has been connected to the regulation of p53 through multiple mechanisms.
E2Fl protein levels are dramatically stabilized following different types of DNA damage
including UV-irradation and gamma-irradiation (Blattner et al., 1999; Hofferer et al.,
1999; Liu et al., 2003; Wang et al., 2004a). This is also associated with increased DNA
binding activity and this property appears to be unique among the E2Fs. The induction of
41
E2F1 following DNA damage appears to depend upon the ATM kinase. Both ATM and
Chk2 are able to phosphorylate E2F1 in response to DNA damage (Lin et al., 2001;
Stevens et al., 2003). Additionally, E2F has been shown to regulate the expression of
both ATM and Chk2 and E2F1 is able to indirectly cause the phosphorylation of p53
through the activity of these kinases (Berkovich and Ginsberg, 2003; Powers et al., 2004;
Rogoff et al., 2002; Rogoff et al., 2004). Several E2F target genes have also been
recently identified with roles in apoptosis. E2F can induce the transcription of genes
such as Apaf-1, p73, caspase 7, pl9Arf, Puma, Noxa, and Bim (Hershko and Ginsberg,
2004; Irwin et al., 2000; Moroni et al., 2001; Pediconi et al., 2003; Stiewe and Putzer,
2000). E2F1 has also been reported to repress expression of the anti-apoptotic gene Bc12
(Eischen et al., 2001). Additionally, it has been demonstrated that E2F can regulate the
expression of p53 co-factors ASPP1, ASPP2, JMY, and TP53INP1 (Hershko et al.,
2005). It has been suggested that the connection between E2F and p53 represents a
mechanism by which cells can monitor proliferation levels, and this may be relevant as a
part of the tumor surveillance network. However, these interactions remain to be
clarified in mouse models.
Role of the p53 in transformation and tumorigenesis
Mouse models have helped address the role of p53 in tumorigenesis and assess
the involvement of other components of the p53 pathway in this process. In particular,
the role of p19ARF has been of interest as a positive upstream regulator of p53 in the
tumor surveillance network.
p53 is mostly dispensable for development although a subtle female-specific
lethality is seen due to failure to close the neural tube. The reason for this is not known.
42
Expression of p19ARF is not detectable in most mouse tissues during development and in
young mice (Zindy et al., 1997). One exception is in the eye, where p19ARF has been
shown to play an essential role in hyaloid vascular regression (McKeller et al., 2002;
Silva et al., 2005). Consequently, pl9Arf knockout mice are blind. This function is not
shared by p53. There is also evidence to suggest that pl9Arf has a role in the mammary
gland of mice (Foster and Lozano, 2002; Yi et al., 2004). Finally, a recent study inserted
GFP into the endogenous pl9Arf locus and was able to observe that spontaneous tumors
arising in these mice were GFP-positive (Zindy et al., 2003). This study demonstrated
that p19ARF expression is specifically induced during tumorigenesis.
p53-deficient mice are prone to the development of spontaneous tumors, mainly
thymic lymphomas and soft tissue sarcomas (Attardi and Jacks, 1999). pl9Arf knockout
mice develop spontaneous tumors that include mostly lymphomas and soft tissue
sarcomas, as well as some tumors of neuronal origin (Kamijo et al., 1999; Kamijo et al.,
1997). The tumor spectrum of these mice is similar but not the same as that seen in p53
knockout mice, suggesting that these two genes possess both common and independent
functions (Moore et al., 2003; Weber et al., 2000). Both p53 and pl9Arfknockout
mice
are also highly susceptible to carcinogen-induced tumors. Finally, p53 and p19Arf are
often spontaneously inactivated in other mouse tumor models (Baudino et al., 2003).
There is evidence that the loss of pl9Arf and p53 are not completely identical in
tumorigenesis. Direct comparision of these genotypes in MEF transformation studies
demonstrated that p53-deficiency is more tumorigenic than pl9Arf-deficiency (Sharpless
et al., 2004). The difference between p53-loss and p19Arf-loss was further demonstrated
by experiments showing that pl9Arf;mdm2;p53 triple knockout mice succumb to tumors
43
more rapidly than mdm2;p53 double knockout mice (Moore et al., 2003). This could also
reflect tissue-specific differences in expression.
Crosses with other mouse tumor models have also highlighted differences
between p53 and pl9ARF. p53-deficiency
but notpl9Arf-deficiency
cooperates with
Patched (Ptc) heterozygosity in the development of medulloblastoma (Wetmore et al.,
2001). This has been attributed to the effect of p53 on genome stability. In Rb
heterozygous mice, the p19Arf-deficiency, but not p53-deficiency, accelerates pituitary
tumor development (Tsai et al., 2002b; Williams et al., 1994a). In contrast, p53-loss but
not p19Arf-loss cooperates with Rb heterozygosity in the development of novel tumor
types. Bax and Nfl mutant mice have also been crossed with both p53 and pl9Arf mutant
mice (Cichowski et al., 1999; Eischen et al., 2002; King et al., 2002; Knudson et al.,
2001; Reilly et al., 2000). These studies all clearly demonstrate that the loss of either p53
or pl9Arfis not always equivalent in tumorigenesis.
In the following chapters, I will examine the crosstalk between E2F3 and the p53
pathway during cell cycle re-entry, asynchronous proliferation, development and
tumorigenesis.
To this end, I have generated both E2f3,;p9Arf and E2f3;p53 compound
mutant cells and mice. In Chapter 2, I examine the regulation of the p53 pathway during
cell cycle re-entry through E2F3-mediated repression of pl9Arf
In Chapter 3, I examine
the relationship between p19ARF and E2F3 in the context of cellular proliferation,
development and tumorigenesis.
In Chapter 4, I examine the relationship between p53
and E2F3 in the context of cellular proliferation, development and tumorigenesis.
I also
assess whether p19ARF and p53 function differently in these processes. Together, these
44
studies have established that E2F3 plays a key role in regulating the cell cycle
progression through both pl9Arf-dependent and pl9Arf-independent mechanisms.
45
References.
Adams, M. R., Sears, R., Nuckolls, F., Leone, G., and Nevins, J. R. (2000). Complex
transcriptional regulatory mechanisms control expression of the E2F3 locus. Mol Cell
Biol 20, 3633-3639.
Alexander, K., and Hinds, P. W. (2001). Requirement for p27(KIP1) in retinoblastoma
protein-mediated senescence. Mol Cell Biol 21, 3616-3631.
Allen, K. E., de la Luna, S., Kerkhoven, R. M., Bernards, R., and La Thangue, N. B.
(1997). Distinct mechanisms of nuclear accumulation regulate the functional
consequence of E2F transcription factors. J Cell Sci 110 ( Pt 22), 2819-2831.
Ameyar-Zazoua, M., Wisniewska, M. B., Bakiri, L., Wagner, E. F., Yaniv, M., and
Weitzman, J. B. (2005). AP-1 dimers regulate transcription of the p 14/p19ARF tumor
suppressor gene. Oncogene 24, 2298-2306.
Appella, E., and Anderson, C. W. (2001). Post-translational modifications and activation
of p53 by genotoxic stresses. Eur J Biochem 268, 2764-2772.
Attardi, L. D., and Jacks, T. (1999). The role of p53 in tumour suppression: lessons from
mouse models. Cell Mol Life Sci 55, 48-63.
Attardi, L. D., Reczek, E. E., Cosmas, C., Demicco, E. G., McCurrach, M. E., Lowe, S.
W., and Jacks, T. (2000). PERP, an apoptosis-associated target of p53, is a novel member
of the PMP-22/gas3 family. Genes Dev 14, 704-718.
Attwooll, C., Oddi, S., Cartwright, P., Prosperini, E., Agger, K., Steensgaard, P.,
Wagener, C., Sardet, C., Moroni, M. C., and Helin, K. (2005). A novel repressive E2F6
complex containing the polycomb group protein, EPC1, that interacts with EZH2 in a
proliferation-specific
manner. J Biol Chem 280, 1199-1208.
Bagui, T. K., Mohapatra, S., Haura, E., and Pledger, W. J. (2003). P27Kipl and p21Cipl
are not required for the formation of active D cyclin-cdk4 complexes. Mol Cell Biol 23,
7285-7290.
Banin, S., Moyal, L., Shieh, S., Taya, Y., Anderson, C. W., Chessa, L., Smorodinsky, N.
I., Prives, C., Reiss, Y., Shiloh, Y., and Ziv, Y. (1998). Enhanced phosphorylation of p53
by ATM in response to DNA damage. Science 281, 1674-1677.
Barlow, C., Hirotsune, S., Paylor, R., Liyanage, M., Eckhaus, M., Collins, F., Shiloh, Y.,
Crawley, J. N., Ried, T., Tagle, D., and Wynshaw-Boris, A. (1996). Atm-deficient mice:
a paradigm of ataxia telangiectasia. Cell 86, 159-171.
Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden,
K. H. (1998). p14ARF links the tumour suppressors RB and p53. Nature 395, 124-125.
46
Baudino, T. A., Maclean, K. H., Brennan, J., Parganas, E., Yang, C., Aslanian, A., Lees,
J. A., Sherr, C. J., Roussel, M. F., and Cleveland, J. L. (2003). Myc-mediated
proliferation and lymphomagenesis, but not apoptosis, are compromised by E2fl loss.
Mol Cell 11, 905-914.
Bell, D. W., Varley, J. M., Szydlo, T. E., Kang, D. H., Wahrer, D. C., Shannon, K. E.,
Lubratovich, M., Verselis, S. J., Isselbacher, K. J., Fraumeni, J. F., et al. (1999).
Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome. Science 286, 25282531.
Berkovich, E., and Ginsberg, D. (2003). ATM is a target for positive regulation by E2F1. Oncogene 22, 161-167.
Berthet, C., Aleem, E., Coppola, V., Tessarollo, L., and Kaldis, P. (2003). Cdk2 knockout
mice are viable. Curr Biol 13, 1775-1785.
Bertwistle, D., Sugimoto, M., and Sherr, C. J. (2004). Physical and functional interactions
of the Arf tumor suppressor protein with nucleophosmin/B23. Mol Cell Biol 24, 985-996.
Bharadwaj, R., and Yu, H. (2004). The spindle checkpoint, aneuploidy, and cancer.
Oncogene 23, 2016-2027.
Bhattacharya, S., Garriga, J., Calbo, J., Yong, T., Haines, D. S., and Grana, X. (2003).
SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human
cells. Oncogene 22, 2443-2451.
Blasco, M. A., Lee, H. W., Rizen, M., Hanahan, D., DePinho, R., and Greider, C. W.
(1997). Mouse models for the study of telomerase. Ciba Found Symp 211, 160-170;
discussion 170-166.
Blattner, C., Sparks, A., and Lane, D. (1999). Transcription factor E2F-1 is upregulated
in response to DNA damage in a manner analogous to that of p53. Mol Cell Biol 19,
3704-3713.
Bode, A. M., and Dong, Z. (2004). Post-translational modification of p53 in
tumorigenesis. Nat Rev Cancer 4, 793-805.
Brehm, A., Miska, E. A., McCance, D. J., Reid, J. L., Bannister, A. J., and Kouzarides, T.
(1998). Retinoblastoma protein recruits histone deacetylase to repress transcription.
Nature 391, 597-601.
Brugarolas, J., Chandrasekaran, C., Gordon, J. I., Beach, D., Jacks, T., and Hannon, G. J.
(1995). Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377,
552-557.
Bruins, W., Zwart, E., Attardi, L. D., Iwakuma, T., Hoogervorst, E. M., Beems, R. B.,
Miranda, B., van Oostrom, C. T., van den Berg, J., van den Aardweg, G. J., et al. (2004).
47
Increased sensitivity to UV radiation in mice with a p53 point mutation at Ser389. Mol
Cell Biol 24, 8884-8894.
Brummelkamp, T. R., Kortlever, R. M., Lingbeek, M., Trettel, F., MacDonald, M. E., van
Lohuizen, M., and Bernards, R. (2002). TBX-3, the gene mutated in Ulnar-Mammary
Syndrome, is a negative regulator of p19ARF and inhibits senescence. J Biol Chem 277,
6567-6572.
Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J. P., Sedivy, J. M.,
Kinzler, K. W., and Vogelstein, B. (1998). Requirement for p53 and p21 to sustain G2
arrest after DNA damage. Science 282, 1497-1501.
Callaghan, D. A., Dong, L., Callaghan, S. M., Hou, Y. X., Dagnino, L., and Slack, R. S.
(1999). Neural precursor cells differentiating in the absence of Rb exhibit delayed
terminal mitosis and deregulated E2F 1 and 3 activity. Dev Biol 207, 257-270.
Campanero, M. R., and Flemington, E. K. (1997). Regulation of E2F through ubiquitinproteasome-dependent degradation: stabilization by the pRB tumor suppressor protein.
Proc Natl Acad Sci U S A 94, 2221-2226.
Canman, C. E., Lim, D. S., Cimprich, K. A., Taya, Y., Tamai, K., Sakaguchi, K.,
Appella, E., Kastan, M. B., and Siliciano, J. D. (1998). Activation of the ATM kinase by
ionizing radiation and phosphorylation of p53. Science 281, 1677-1679.
Cartwright, P., Muller, H., Wagener, C., Holm, K., and Helin, K. (1998). E2F-6: a novel
member of the E2F family is an inhibitor of E2F-dependent transcription. Oncogene 17,
611-623.
Castano, E., Kleyner, Y., and Dynlacht, B. D. (1998). Dual cyclin-binding domains are
required for p107 to function as a kinase inhibitor. Mol Cell Biol 18, 5380-5391.
Ceol, C. J., and Horvitz, H. R. (2001). dpl-1 DP and efl-1 E2F act with lin-35 Rb to
antagonize Ras signaling in C. elegans vulval development. Mol Cell 7, 461-473.
Chang, C., Simmons, D. T., Martin, M. A., and Mora, P. T. (1979). Identification and
partial characterization of new antigens from simian virus 40-transformed mouse cells. J
Virol 31, 463-471.
Chao, C., Hergenhahn, M., Kaeser, M. D., Wu, Z., Saito, S., Iggo, R., Hollstein, M.,
Appella, E., and Xu, Y. (2003). Cell type- and promoter-specific roles of Serl8
phosphorylation in regulating p53 responses. J Biol Chem 278, 41028-41033.
Chehab, N. H., Malikzay, A., Appel, M., and Halazonetis, T. D. (2000). Chk2/hCdsl
functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev 14, 278288.
48
Chehab, N. H., Malikzay, A., Stavridi, E. S., and Halazonetis, T. D. (1999).
Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA
damage. Proc Natl Acad Sci U S A 96, 13777-13782.
Chen, D., Livne-bar, I., Vanderluit, J. L., Slack, R. S., Agochiya, M., and Bremner, R.
(2004). Cell-specific effects of RB or RB/p107 loss on retinal development implicate an
intrinsically death-resistant cell-of-origin in retinoblastoma. Cancer Cell 5, 539-551.
C(hen, G., and Lee, E. Y. (1999). Phenotypic differentiation without permanent cell-cycle
arrest by skeletal myocytes with deregulated E2F-1. DNA Cell Biol 18, 305-314.
Chen, Z., Trotman, L. C., Shaffer, D., Lin, H. K., Dotan, Z. A., Niki, M., Koutcher, J. A.,
Scher, H. I., Ludwig, T., Gerald, W., et al. (2005). Crucial role of p53-dependent cellular
senescence in suppression of Pten-deficient tumorigenesis. Nature 436, 725-730.
Cheng, M., Olivier, P., Diehl, J. A., Fero, M., Roussel, M. F., Roberts, J. M., and Sherr,
C. J. (1999). The p21(Cipl) and p27(Kipl) CDK 'inhibitors' are essential activators of
cyclin D-dependent kinases in murine fibroblasts. Embo J 18, 1571-1583.
Chibazakura, T., McGrew, S. G., Cooper, J. A., Yoshikawa, H., and Roberts, J. M.
(2004). Regulation of cyclin-dependent kinase activity during mitotic exit and
maintenance of genome stability by p21, p27, and p107. Proc Natl Acad Sci U S A 101,
4465-4470.
Chipuk, J. E., Kuwana, T., Bouchier-Hayes, L., Droin, N. M., Newmeyer, D. D., Schuler,
M., and Green, D. R. (2004). Direct activation of Bax by p53 mediates mitochondrial
membrane permeabilization and apoptosis. Science 303, 1010-1014.
Cho, Y., Gorina, S., Jeffrey, P. D., and Pavletich, N. P. (1994). Crystal structure of a p53
tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265,
346-355.
Cichowski, K., Shih, T. S., Schmitt, E., Santiago, S., Reilly, K., McLaughlin, M. E.,
Bronson, R. T., and Jacks, T. (1999). Mouse models of tumor development in
neurofibromatosis type 1. Science 286, 2172-2176.
Clarke, A. R., Maandag, E. R., van Roon, M., van der Lugt, N. M., van der Valk, M.,
Hooper, M. L., Berns, A., and te Riele, H. (1992). Requirement for a functional Rb-1
gene in murine development. Nature 359, 328-330.
Classon, M., and Harlow, E. (2002). The retinoblastoma tumour suppressor in
development and cancer. Nat Rev Cancer 2, 910-917.
Cloud, J. E., Rogers, C., Reza, T. L., Ziebold, U., Stone, J. R., Picard, M. H., Caron, A.
M., Bronson, R. T., and Lees, J. A. (2002). Mutant mouse models reveal the relative roles
of E2F1 and E2F3 in vivo. Mol Cell Biol 22, 2663-2672.
49
Courtois, S., de Fromentel, C. C., and Hainaut, P. (2004). p53 protein variants: structural
and functional similarities with p63 and p73 isoforms. Oncogene 23, 631-638.
Coverley, D., Laman, H., and Laskey, R. A. (2002). Distinct roles for cyclins E and A
during DNA replication complex assembly and activation. Nat Cell Biol 4, 523-528.
Dannenberg, J. H., van Rossum, A., Schuijff, L., and te Riele, H. (2000). Ablation of the
retinoblastoma gene family deregulates G(1) control causing immortalization and
increased cell turnover under growth-restricting conditions. Genes Dev 14, 3051-3064.
Datar, S. A., Jacobs, H. W., de la Cruz, A. F., Lehner, C. F., and Edgar, B. A. (2000). The
Drosophila cyclin D-Cdk4 complex promotes cellular growth. Embo J 19, 4543-4554.
de Bruin, A., Maiti, B., Jakoi, L., Timmers, C., Buerki, R., and Leone, G. (2003).
Identification and characterization of E2F7, a novel mammalian E2F family member
capable of blocking cellular proliferation. J Biol Chem 278, 42041-42049.
de la Luna, S., Burden, M. J., Lee, C. W., and La Thangue, N. B. (1996). Nuclear
accumulation of the E2F heterodimer regulated by subunit composition and alternative
splicing of a nuclear localization signal. J Cell Sci 109 ( Pt 10), 2443-2452.
de Stanchina, E., McCurrach, M. E., Zindy, F., Shieh, S. Y., Ferbeyre, G., Samuelson, A.
V., Prives, C., Roussel, M. F., Sherr, C. J., and Lowe, S. W. (1998). E1A signaling to p53
involves the p19(ARF) tumor suppressor. Genes Dev 12, 2434-2442.
DeGregori, J., Leone, G., Miron, A., Jakoi, L., and Nevins, J. R. (1997). Distinct roles for
E2F proteins in cell growth control and apoptosis. Proc Natl Acad Sci U S A 94, 72457250.
Denchi, E. L., Attwooll, C., Pasini, D., and Helin, K. (2005). Deregulated E2F activity
induces hyperplasia and senescence-like features in the mouse pituitary gland. Mol Cell
Biol 25, 2660-2672.
Deng, C., Zhang, P., Harper, J. W., Elledge, S. J., and Leder, P. (1995). Mice lacking
p21CIP1/WAF1 undergo normal development, but are defective in GI checkpoint
control. Cell 82, 675-684.
Di Como, C. J., Gaiddon, C., and Prives, C. (1999). p7 3 function is inhibited by tumorderived p53 mutants in mammalian cells. Mol Cell Biol 19, 1438-1449.
Di Stefano, L., Jensen, M. R., and Helin, K. (2003). E2F7, a novel E2F featuring DP-
independent repression of a subset of E2F-regulated genes. Embo J 22, 6289-6298.
Dimova, D. K., Stevaux, O., Frolov, M. V., and Dyson, N. J. (2003). Cell cycledependent and cell cycle-independent control of transcription by the Drosophila E2F/RB
pathway. Genes Dev 17, 2308-2320.
50
Dimri, G. P., Itahana, K., Acosta, M., and Campisi, J. (2000). Regulation of a senescence
checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor.
Mol Cell Biol 20, 273-285.
Dynlacht, B. D., Flores, O., Lees, J. A., and Harlow, E. (1994). Differential regulation of
E2F transactivation by cyclin/cdk2 complexes. Genes Dev 8, 1772-1786.
Dynlacht, B. D., Moberg, K., Lees, J. A., Harlow, E., and Zhu, L. (1997). Specific
regulation of E2F family members by cyclin-dependent kinases. Mol Cell Biol 17, 38673875.
Dyson, N., Guida, P., Munger, K., and Harlow, E. (1992). Homologous sequences in
adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same
set of cellular proteins. J Virol 66, 6893-6902.
Dyson, N., and Harlow, E. (1992). Adenovirus E1A targets key regulators of cell
proliferation. Cancer Surv 12, 161-195.
Edwards, S. J., Hananeia, L., Eccles, M. R., Zhang, Y. F., and Braithwaite, A. W. (2003).
The proline-rich region of mouse p53 influences transactivation and apoptosis but is
largely dispensable for these functions. Oncogene 22, 4517-4523.
Eischen, C. M., Packham, G., Nip, J., Fee, B. E., Hiebert, S. W., Zambetti, G. P., and
Cleveland, J. L. (2001). Bcl-2 is an apoptotic target suppressed by both c-Myc and E2F-
1. Oncogene 20, 6983-6993.
Eischen, C. M., Rehg, J. E., Korsmeyer, S. J., and Cleveland, J. L. (2002). Loss of Bax
alters tumor spectrum and tumor numbers in ARF-deficient mice. Cancer Res 62, 21842191.
el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., and Vogelstein, B. (1992).
Definition of a consensus binding site for p53. Nat Genet 1, 45-49.
el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin,
D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993). WAF1, a potential
mediator of p53 tumor suppression. Cell 75, 817-825.
Ewen, M. E., Xing, Y. G., Lawrence, J. B., and Livingston, D. M. (1991). Molecular
cloning, chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma
gene product-related protein. Cell 66, 1155-1164.
Fajas, L., Landsberg, R. L., Huss-Garcia, Y., Sardet, C., Lees, J. A., and Auwerx, J.
(2002). E2Fs regulate adipocyte differentiation. Dev Cell 3, 39-49.
Feber, A., Clark, J., Goodwin, G., Dodson, A. R., Smith, P. H., Fletcher, A., Edwards, S.,
Flohr, P., Falconer, A., Roe, T., et al. (2004). Amplification and overexpression of E2F3
in human bladder cancer. Oncogene 23, 1627-1630.
51
Ferreira, R., Magnaghi-Jaulin, L., Robin, P., Harel-Bellan, A., and Trouche, D. (1998).
The three members of the pocket proteins family share the ability to repress E2F activity
through recruitment of a histone deacetylase. Proc Natl Acad Sci U S A 95, 1049310498.
Fields, S., and Jang, S. K. (1990). Presence of a potent transcription activating sequence
in the p53 protein. Science 249, 1046-1049.
Fletcher, O., Easton, D., Anderson, K., Gilham, C., Jay, M., and Peto, J. (2004). Lifetime
risks of common cancers among retinoblastoma survivors. J Natl Cancer Inst 96, 357363.
Flores, E. R., Sengupta, S., Miller, J. B., Newman, J. J., Bronson, R., Crowley, D., Yang,
A., McKeon, F., and Jacks, T. (2005). Tumor predisposition in mice mutant for p63 and
p73: evidence for broader tumor suppressor functions for the p53 family. Cancer Cell 7,
363-373.
Flores, E. R., Tsai, K. Y., Crowley, D., Sengupta, S., Yang, A., McKeon, F., and Jacks,
T. (2002). p 6 3 and p73 are required for p53-dependent apoptosis in response to DNA
damage. Nature 416, 560-564.
Foster, C. J., and Lozano, G. (2002). Loss of p19ARF enhances the defects of Mdm2
overexpression in the mammary gland. Oncogene 21, 3525-3531.
Foster, C. S., Falconer, A., Dodson, A. R., Norman, A. R., Dennis, N., Fletcher, A.,
Southgate, C., Dowe, A., Dearnaley, D., Jhavar, S., et al. (2004). Transcription factor
E2F3 overexpressed in prostate cancer independently predicts clinical outcome.
Oncogene 23, 5871-5879.
Fridman, J. S., and Lowe, S. W. (2003). Control of apoptosis by p53. Oncogene 22,
9030-9040.
Friedman, P. N., Chen, X., Bargonetti, J., and Prives, C. (1993). The p53 protein is an
unusually shaped tetramer that binds directly to DNA. Proc Natl Acad Sci U S A 90,
3319-3323.
Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M., Albert, D. M.,
and Dryja, T. P. (1986). A human DNA segment with properties of the gene that
predisposes to retinoblastoma and osteosarcoma. Nature 323, 643-646.
Frolov, M. V., Huen, D. S., Stevaux, O., Dimova, D., Balczarek-Strang, K., Elsdon, M.,
and Dyson, N. J. (2001). Functional antagonism between E2F family members. Genes
Dev 15, 2146-2160.
Fung, Y. K., Murphree, A. L., T'Ang, A., Qian, J., Hinrichs, S. H., and Benedict, W. F.
(1987). Structural evidence for the authenticity of the human retinoblastoma gene.
Science 236, 1657-1661.
52
Gad, A., Thullberg, M., Dannenberg, J. H., te Riele, H., and Stromblad, S. (2004).
Retinoblastoma susceptibility gene product (pRb) and p107 functionally separate the
requirements for serum and anchorage in the cell cycle GI-phase. J Biol Chem 279,
13640-13644.
Gaiddon, C., Lokshin, M., Ahn, J., Zhang, T., and Prives, C. (2001). A subset of tumor-
derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with
the p53 core domain. Mol Cell Biol 21, 1874-1887.
Galbiati, L., Mendoza-Maldonado,
R., Gutierrez, M. I., and Giacca, M. (2005).
Regulation of E2F-1 After DNA Damage by p300-Mediated Acetylation and
Ubiquitination. Cell Cycle 4.
Gaubatz, S., Lees, J. A., Lindeman, G. J., and Livingston, D. M. (2001). E2F4 is exported
from the nucleus in a CRM1-dependent manner. Mol Cell Biol 21, 1384-1392.
Gaubatz, S., Lindeman, G. J., Ishida, S., Jakoi, L., Nevins, J. R., Livingston, D. M., and
Rempel, R. E. (2000). E2F4 and E2F5 play an essential role in pocket protein-mediated
G1 control. Mol Cell 6, 729-735.
Geng, Y., Whoriskey, W., Park, M. Y., Bronson, R. T., Medema, R. H., Li, T., Weinberg,
R. A., and Sicinski, P. (1999). Rescue of cyclin D1 deficiency by knockin cyclin E. Cell
97, 767-777.
Geng, Y., Yu, Q., Sicinska, E., Das, M., Schneider, J. E., Bhattacharya, S., Rideout, W.
M., Bronson, R. T., Gardner, H., and Sicinski, P. (2003). Cyclin E ablation in the mouse.
Cell 114, 431-443.
Giangrande, P. H., Hallstrom, T. C., Tunyaplin, C., Calame, K., and Nevins, J. R. (2003).
Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription
factor. Mol Cell Biol 23, 3707-3720.
Giangrande, P. H., Zhu, W., Rempel, R. E., Laakso, N., and Nevins, J. R. (2004).
Combinatorial gene control involving E2F and E Box family members. Embo J 23, 13361347.
Gille, H., and Downward, J. (1999). Multiple ras effector pathways contribute to G(1)
cell cycle progression. J Biol Chem 274, 22033-22040.
Girling, R., Partridge, J. F., Bandara, L. R., Burden, N., Totty, N. F., Hsuan, J. J., and La
Thangue, N. B. (1993). A new component of the transcription factor DRTF1/E2F. Nature
365, 468.
Guo, F., Gao, Y., Wang, L., and Zheng, Y. (2003). pl9Arf-p53 tumor suppressor
pathway regulates cell motility by suppression of phosphoinositide 3-kinase and Racl
GTPase activities. J Biol Chem 278, 14414-14419.
53
Guo, F., and Zheng, Y. (2004). Involvement of Rho family GTPases in pl9Arf- and p53-
mediated proliferation of primary mouse embryonic fibroblasts. Mol Cell Biol 24, 14261438.
Gutierrez, C., Ramirez-Parra, E., Castellano, M. M., and del Pozo, J. C. (2002). G(1) to S
transition: more than a cell cycle engine switch. Curr Opin Plant Biol 5, 480-486.
Hallstrom, T. C., and Nevins, J. R. (2003). Specificity in the activation and control of
transcription factor E2F-dependent apoptosis. Proc Natl Acad Sci U S A 100, 1084810853.
Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.
Hansen, D. V., Hsu, J. Y., Kaiser, B. K., Jackson, P. K., and Eldridge, A. G. (2002).
Control of the centriole and centrosome cycles by ubiquitination enzymes. Oncogene 21,
6209-6221.
Harley, C. B., Futcher, A. B., and Greider, C. W. (1990). Telomeres shorten during
ageing of human fibroblasts. Nature 345, 458-460.
Hasan, M. K., Yaguchi, T., Sugihara, T., Kumar, P. K., Taira, K., Reddel, R. R., Kaul, S.
C., and Wadhwa, R. (2002). CARF is a novel protein that cooperates with mouse
p19ARF (human p14ARF) in activating p53. J Biol Chem 277, 37765-37770.
Hateboer, G., Kerkhoven, R. M., Shvarts, A., Bernards, R., and Beijersbergen, R. L.
(1996). Degradation of E2F by the ubiquitin-proteasome pathway: regulation by
retinoblastoma family proteins and adenovirus transforming proteins. Genes Dev 10,
2960-2970.
Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997). Mdm2 promotes the rapid
degradation of p53. Nature 387, 296-299.
Hayflick, L. (1965). The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp
Cell Res 37, 614-636.
Hayflick, L., and Moorhead, P. S. (1961). The serial cultivation of human diploid cell
strains. Exp Cell Res 25, 585-621.
Helin, K., Harlow, E., and Fattaey, A. (1993a). Inhibition of E2F-1 transactivation by
direct binding of the retinoblastoma protein. Mol Cell Biol 13, 6501-6508.
Helin, K., Lees, J. A., Vidal, M., Dyson, N., Harlow, E., and Fattaey, A. (1992). A cDNA
encoding a pRB-binding protein with properties of the transcription factor E2F. Cell 70,
337-350.
Helin, K., Wu, C. L., Fattaey, A. R., Lees, J. A., Dynlacht, B. D., Ngwu, C., and Harlow,
E. (1993b). Heterodimerization of the transcription factors E2F-1 and DP-1 leads to
cooperative trans-activation. Genes Dev 7, 1850-1861.
54
Hermeking, H., Lengauer, C., Polyak, K., He, T. C., Zhang, L., Thiagalingam, S.,
Kinzler, K. W., and Vogelstein, B. (1997). 14-3-3 sigma is a p53-regulated inhibitor of
G2/M progression. Mol Cell 1, 3-11.
Hernando, E., Nahle, Z., Juan, G., Diaz-Rodriguez, E., Alaminos, M., Hemann, M.,
Michel, L., Mittal, V., Gerald, W., Benezra, R., et al. (2004). Rb inactivation promotes
genomic instability by uncoupling cell cycle progression from mitotic control. Nature
430, 797-802.
Hershko, T., Chaussepied, M., Oren, M., and Ginsberg, D. (2005). Novel link between
E2F and p53: proapoptotic cofactors of p53 are transcriptionally upregulated by E2F.
Cell Death Differ 12, 377-383.
Hershko, T., and Ginsberg, D. (2004). Up-regulation of Bcl-2 homology 3 (BH3)-only
proteins by E2F1 mediates apoptosis. J Biol Chem 279, 8627-8634.
Hiebert, S. W., Blake, M., Azizkhan, J., and Nevins, J. R. (1991). Role of E2F
transcription factor in EIA-mediated trans activation of cellular genes. J Virol 65, 35473552.
Hirao, A., Kong, Y. Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida, H., Liu, D.,
Elledge, S. J., and Mak, T. W. (2000). DNA damage-induced activation of p53 by the
checkpoint kinase Chk2. Science 287, 1824-1827.
Hofferer, M., Wirbelauer, C., Humar, B., and Krek, W. (1999). Increased levels of E2F1-dependent I)NA binding activity after UV- or gamma-irradiation. Nucleic Acids Res
27, 491-495.
Hofmann, F., Martelli, F., Livingston, D. M., and Wang, Z. (1996). The retinoblastoma
gene product protects E2F-1 from degradation by the ubiquitin-proteasome pathway.
Genes Dev 10, 2949-2959.
Honda, R., Tanaka, H., and Yasuda, H. (1997). Oncoprotein MDM2 is a ubiquitin ligase
E3 for tumor suppressor p53. FEBS Lett 420, 25-27.
Honda, R., and Yasuda, H. (1999). Association of p19(ARF) with Mdm2 inhibits
ubiquitin ligase activity of Mdm2 for tumor suppressor p53. Embo J 18, 22-27.
Hoogervorst, E. M., Bruins, W., Zwart, E., van Oostrom, C. T., van den Aardweg, G. J.,
Beems, R. B., van den Berg, J., Jacks, T., van Steeg, H., and de Vries, A. (2005). Lack of
p53 Ser389 phosphorylation predisposes mice to develop 2-acetylaminofluorene-induced
bladder tumors but not ionizing radiation-induced lymphomas. Cancer Res 65, 36103616.
Hsiao, K. M., McMahon, S. L., and Farnham, P. J. (1994). Multiple DNA elements are
required for the growth regulation of the mouse E2F1 promoter. Genes Dev 8, 15261537.
55
Hsieh, J. K., Chan, F. S., O'Connor, D. J., Mittnacht, S., Zhong, S., and Lu, X. (1999).
RB regulates the stability and the apoptotic function of p53 via MDM2. Mol Cell 3, 181193.
Huang, E., Ishida, S., Pittman, J., Dressman, H., Bild, A., Kloos, M., D'Amico, M.,
Pestell, R. G., West, M., and Nevins, J. R. (2003). Gene expression phenotypic models
that predict the activity of oncogenic pathways. Nat Genet 34, 226-230.
Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1991). A cellular protein mediates
association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18.
Embo J 10, 4129-4135.
Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1993). Cloning and expression of
the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus
E6 oncoprotein with p53. Mol Cell Biol 13, 775-784.
Humbert, P. O., Rogers, C., Ganiatsas, S., Landsberg, R. L., Trimarchi, J. M., Dandapani,
S., Brugnara, C., Erdman, S., Schrenzel, M., Bronson, R. T., and Lees, J. A. (2000a).
E2F4 is essential for normal erythrocyte maturation and neonatal viability. Mol Cell 6,
281-291.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000b). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
Hurford, R. K., Jr., Cobrinik, D., Lee, M. H., and Dyson, N. (1997). pRB and p107/p130
are required for the regulated expression of different sets of E2F responsive genes. Genes
Dev 11, 1447-1463.
Iliakis, G., Wang, Y., Guan, J., and Wang, H. (2003). DNA damage checkpoint control in
cells exposed to ionizing radiation. Oncogene 22, 5834-5847.
Irwin, M., Marin, M. C., Phillips, A. C., Seelan, R. S., Smith, D. I., Liu, W., Flores, E. R.,
Tsai, K. Y., Jacks, T., Vousden, K. H., and Kaelin, W. G., Jr. (2000). Role for the p53
homologue p73 in E2F-1-induced apoptosis. Nature 407, 645-648.
Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M., and Nevins, J. R.
(2001). Role for E2F in control of both DNA replication and mitotic functions as
revealed from DNA microarray analysis. Mol Cell Biol 21, 4684-4699.
Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, M. A., and Weinberg, R.
A. (1992). Effects of an Rb mutation in the mouse. Nature 359, 295-300.
Jacobs, J. J., Keblusek, P., Robanus-Maandag, E., Kristel, P., Lingbeek, M., Nederlof, P.
M., van Welsem, T., van de Vijver, M. J., Koh, E. Y., Daley, G. Q., and van Lohuizen,
M. (2000). Senescence bypass screen identifies TBX2, which represses Cdkn2a
(p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet 26, 291-299.
56
Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A., and van Lohuizen, M. (1999).
The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and
senescence through the ink4a locus. Nature 397, 164-168.
Jeffrey, P. D., Gorina, S., and Pavletich, N. P. (1995). Crystal structure of the
tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science 267, 14981502.
Jenkins, J. R., Rudge, K., Chumakov, P., and Currie, G. A. (1985). The cellular oncogene
p53 can be activated by mutagenesis. Nature 317, 816-818.
Jones, S. N., Roe, A. E., Donehower, L. A., and Bradley, A. (1995). Rescue of embryonic
lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208.
Jost, C. A., Ginsberg, D., and Kaelin, W. G., Jr. (1996). A conserved region of unknown
function participates in the recognition of E2F family members by the adenovirus E4
ORF 6/7 protein. Virology 220, 78-90.
Kalinichenko, V'. V., Major, M. L., Wang, X., Petrovic, V., Kuechle, J., Yoder, H. M.,
Dennewitz, M. B., Shin, B., Datta, A., Raychaudhuri, P., and Costa, R. H. (2004).
Foxmlb transcription factor is essential for development of hepatocellular carcinomas
and is negatively regulated by the p19ARF tumor suppressor. Genes Dev 18, 830-850.
Kalma, Y., Marash, L., Lamed, Y., and Ginsberg, D. (2001). Expression analysis using
DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication
genes including replication protein A2. Oncogene 20, 1379-1387.
Kamijo, T., van de Kamp, E., Chong, M. J., Zindy, F., Diehl, J. A., Sherr, C. J., and
McKinnon, P. J. (1999). Loss of the ARF tumor suppressor reverses premature
replicative arrest but not radiation hypersensitivity arising from disabled atm function.
Cancer Res 59, 2464-2469.
Kamijo, T., Weber, J. D., Zambetti, G., Zindy, F., Roussel, M. F., and Sherr, C. J. (1998).
Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2.
Proc Natl Acad Sci U S A 95, 8292-8297.
Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing, J. R., Ashmun, R. A.,
Grosveld, G., and Sherr, C. J. (1997). Tumor suppression at the mouse INK4a locus
mediated by the alternative reading frame product p19ARF. Cell 91, 649-659.
Kapoor, M., and Lozano, G. (1998). Functional activation of p53 via phosphorylation
following DNA damage by UV but not gamma radiation. Proc Natl Acad Sci U S A 95,
2834-2837.
Kastan, M. B., and Bartek, J. (2004). Cell-cycle checkpoints and cancer. Nature 432, 316323.
57
Kel, A. E., Kel-Margoulis, O. V., Farnham, P. J., Bartley, S. M., Wingender, E., and
Zhang, M. Q. (2001). Computer-assisted identification of cell cycle-related genes: new
targets for E2F transcription factors. J Mol Biol 309, 99-120.
King, D., Yang, G., Thompson, M. A., and Hiebert, S. W. (2002). Loss of
neurofibromatosis-1 and p19(ARF) cooperate to induce a multiple tumor phenotype.
Oncogene 21, 4978-4982.
Knudsen, E. S., and Wang, J. Y. (1997). Dual mechanisms for the inhibition of E2F
binding to RB by cyclin-dependent kinase-mediated RB phosphorylation. Mol Cell Biol
17, 5771-5783.
Knudson, C. M., Johnson, G. M., Lin, Y., and Korsmeyer, S. J. (2001). Bax accelerates
tumorigenesis in p53-deficient mice. Cancer Res 61, 659-665.
Korgaonkar, C., Hagen, J., Tompkins, V., Frazier, A. A., Allamargot, C., Quelle, F. W.,
and Quelle, D. E. (2005). Nucleophosmin (B23) targets ARF to nucleoli and inhibits its
function. Mol Cell Biol 25, 1258-1271.
Kovesdi, I., Reichel, R., and Nevins, J. R. (1986). Identification of a cellular transcription
factor involved in E1A trans-activation. Cell 45, 219-228.
Kozar, K., Ciemerych, M. A., Rebel, V. I., Shigematsu, H., Zagozdzon, A., Sicinska, E.,
Geng, Y., Yu, Q., Bhattacharya, S., Bronson, R. T., et al. (2004). Mouse development
and cell proliferation in the absence of D-cyclins. Cell 118, 477-491.
Kraiss, S., Quaiser, A., Oren, M., and Montenarh, M. (1988). Oligomerization of
oncoprotein p53. J Virol 62, 4737-4744.
Krek, W., Ewen, M. E., Shirodkar, S., Arany, Z., Kaelin, W. G., Jr., and Livingston, D.
M. (1994). Negative regulation of the growth-promoting transcription factor E2F-1 by a
stably bound cyclin A-dependent protein kinase. Cell 78, 161-172.
Kress, M., May, E., Cassingena, R., and May, P. (1979). Simian virus 40-transformed
cells express new species of proteins precipitable by anti-simian virus 40 tumor serum. J
Virol 31, 472-483.
Kubbutat, M. H., Ludwig, R. L., Ashcroft, M., and Vousden, K. H. (1998). Regulation of
Mdm2-directed degradation by the C terminus of p53. Mol Cell Biol 18, 5690-5698.
Kubbutat, M. H., Ludwig, R. L., Levine, A. J., and Vousden, K. H. (1999). Analysis of
the degradation function of Mdm2. Cell Growth Differ 10, 87-92.
Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan, M. B. (1992). Wild-type p53 is
a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A 89,
7491-7495.
58
Kuo, M. L., den Besten, W., Bertwistle, D., Roussel, M. F., and Sherr, C. J. (2004). N-
terminal polyubiquitination and degradation of the Arf tumor suppressor. Genes Dev 18,
1862-1874.
Kuo, M. L., Duncavage, E. J., Mathew, R., den Besten, W., Pei, D., Naeve, D.,
Yamamoto, T., Cheng, C., Sherr, C. J., and Roussel, M. F. (2003). Arf induces p53dependent and --independent antiproliferative genes. Cancer Res 63, 1046-1053.
Kurokawa, K., Tanaka, T., and Kato, J. (1999). p19ARF prevents G1 cyclin-dependent
kinase activation by interacting with MDM2 and activating p53 in mouse fibroblasts.
Oncogene 18, 2718-2727.
Kusek, J. C., Greene, R. M., Nugent, P., and Pisano, M. M. (2000). Expression of the
E2F family of transcription factors during murine development. Int J Dev Biol 44, 267277.
La Thangue, N. B., and Rigby, P. W. (1987). An adenovirus ElA-like transcription factor
is regulated during the differentiation of murine embryonal carcinoma stem cells. Cell 49,
507-513.
La Thangue, N. B., Thimmappaya, B., and Rigby, P. W. (1990). The embryonal
carcinoma stem cell Ela-like activity involves a differentiation-regulated transcription
factor. Nucleic Acids Res 18, 2929-2938.
Lai, A., Kennedy, B. K., Barbie, D. A., Bertos, N. R., Yang, X. J., Theberge, M. C., Tsai,
S. C., Seto, E., Zhang, Y., Kuzmichev, A., et al. (2001). RBP1 recruits the mSIN3-
histone deacetylase complex to the pocket of retinoblastoma tumor suppressor family
proteins found in limited discrete regions of the nucleus at growth arrest. Mol Cell Biol
21, 2918-2932.
Landsberg, R. L., Sero, J. E., Danielian, P. S., Yuan, T. L., Lee, E. Y., and Lees, J. A.
(2003). The role of E2F4 in adipogenesis is independent of its cell cycle regulatory
activity. Proc Natl Acad Sci U S A 100, 2456-2461.
Lane, D. P., and Crawford, L. V. (1979). T antigen is bound to a host protein in SV40-
transformed cells. Nature 278, 261-263.
Lang, S. E., McMahon, S. B., Cole, M. D., and Hearing, P. (2001). E2F transcriptional
activation requires TRRAP and GCN5 cofactors. J Biol Chem 276, 32627-32634.
Lee, E. Y., Cam, H., Ziebold, U., Rayman, J. B., Lees, J. A., and Dynlacht, B. D. (2002).
E2F4 loss suppresses tumorigenesis in Rb mutant mice. Cancer Cell 2, 463-472.
Lee, E. Y., Chang, C. Y., Hu, N., Wang, Y. C., Lai, C. C., Herrup, K., Lee, W. H., and
Bradley, A. (1992). Mice deficient for Rb are nonviable and show defects in neurogenesis
and haematopoiesis. Nature 359, 288-294.
59
Lee, J. O., Russo, A. A., and Pavletich, N. P. (1998). Structure of the retinoblastoma
tumour-suppressor pocket domain bound to a peptide from HPV E7. Nature 391, 859865.
Lee, W. H., Shew, J. Y., Hong, F. D., Sery, T. W., Donoso, L. A., Young, L. J.,
Bookstein, R., and Lee, E. Y. (1987). The retinoblastoma susceptibility gene encodes a
nuclear phosphoprotein associated with DNA binding activity. Nature 329, 642-645.
Lees, J. A., Saito, M., Vidal, M., Valentine, M., Look, T., Harlow, E., Dyson, N., and
Helin, K. (1993). The retinoblastoma protein binds to a family of E2F transcription
factors. Mol Cell Biol 13, 7813-7825.
Leone, G., DeGregori, J., Yan, Z., Jakoi, L., Ishida, S., Williams, R. S., and Nevins, J. R.
(1998). E2F3 activity is regulated during the cell cycle and is required for the induction
of S phase. Genes Dev 12, 2120-2130.
Leone, G., Nuckolls, F., Ishida, S., Adams, M., Sears, R., Jakoi, L., Miron, A., and
Nevins, J. R. (2000). Identification of a novel E2F3 product suggests a mechanism for
determining specificity of repression by Rb proteins. Mol Cell Biol 20, 3626-3632.
Leone, G., Sears, R., Huang, E., Rempel, R., Nuckolls, F., Park, C. H., Giangrande, P.,
Wu, L., Saavedra, H. I., Field, S. J., et al. (2001). Myc requires distinct E2F activities to
induce S phase and apoptosis. Mol Cell 8, 105-113.
Li, Y., Graham, C., Lacy, S., Duncan, A. M., and Whyte, P. (1993). The adenovirus E1Aassociated 130-kD protein is encoded by a member of the retinoblastoma gene family and
physically interacts with cyclins A and E. Genes Dev 7, 2366-2377.
Li, Y., Wu, D., Chen, B., Ingram, A., He, L., Liu, L., Zhu, D., Kapoor, A., and Tang, D.
(2004). ATM activity contributes to the tumor-suppressing functions of pl4ARF.
Oncogene 23, 7355-7365.
Lin, W. C., Lin, F. T., and Nevins, J. R. (2001). Selective induction of E2F1 in response
to DNA damage,. mediated by ATM-dependent phosphorylation. Genes Dev 15, 18331844.
Lindeman, G. J., Dagnino, L., Gaubatz, S., Xu, Y., Bronson, R. T., Warren, H. B., and
Livingston, D. M. (1998). A specific, nonproliferative role for E2F-5 in choroid plexus
function revealed by gene targeting. Genes Dev 12, 1092-1098.
Lingbeek, M. E., Jacobs, J. J., and van Lohuizen, M. (2002). The T-box repressors TBX2
and TBX3 specifically regulate the tumor suppressor gene p14ARF via a variant T-site in
the initiator. J Biol Chem 277, 26120-26127.
Linzer, D. I., and Levine, A. J. (1979). Characterization of a 54K dalton cellular SV40
tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma
cells. Cell 17, 43-52.
60
Linzer, D. I., Maltzman, W., and Levine, A. J. (1979). The SV40 A gene product is
required for the production of a 54,000 MW cellular tumor antigen. Virology 98, 308318.
Liu, K., Lin, F. T., Ruppert, J. M., and Lin, W. C. (2003). Regulation of E2F1 by BRCT
domain-containing protein TopBP1. Mol Cell Biol 23, 3287-3304.
Loeken, M. R., and Brady, J. (1989). The adenovirus EIIA enhancer. Analysis of
regulatory sequences and changes in binding activity of ATF and EIIF following
adenovirus infection. J Biol Chem 264, 6572-6579.
Logan, N., Delavaine, L., Graham, A., Reilly, C., Wilson, J., Brummelkamp, T. R.,
Hijmans, E. M., Bernards, R., and La Thangue, N. B. (2004). E2F-7: a distinctive E2F
family member with an unusual organization of DNA-binding domains. Oncogene 23,
5138-5150.
Loughran, O., and La Thangue, N. B. (2000). Apoptotic and growth-promoting activity
of E2F modulated by MDM2. Mol Cell Biol 20, 2186-2197.
Lu, H., Taya, Y., Ikeda, M., and Levine, A. J. (1998). Ultraviolet radiation, but not
gamma radiation or etoposide-induced DNA damage, results in the phosphorylation of
the murine p53 protein at serine-389. Proc Natl Acad Sci U S A 95, 6399-6402.
Ma, Y., Croxton, R., Moorer, R. L., Jr., and Cress, W. D. (2002). Identification of novel
E2F1-regulated genes by microarray. Arch Biochem Biophys 399, 212-224.
Macaluso, M., Cinti, C., Russo, G., Russo, A., and Giordano, A. (2003). pRb2/p130E2F4/5-HDAC 1-SUV39H 1-p300 and pRb2/p 130-E2F4/5-HDAC 1-SUV39H 1-DNMT 1
multimolecular complexes mediate the transcription of estrogen receptor-alpha in breast
cancer. Oncogene 22, 3511-3517.
MacPherson, D., Kim, J., Kim, T., Rhee, B. K., Van Oostrom, C. T., DiTullio, R. A.,
Venere, M., Halazonetis, T. D., Bronson, R., De Vries, A., et al. (2004a). Defective
apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23. Embo J 23,
3689-3699.
MacPherson, D., Sage, J., Kim, T., Ho, D., McLaughlin, M. E., and Jacks, T. (2004b).
Cell type-specific effects of Rb deletion in the murine retina. Genes Dev 18, 1681-1694.
Magae, J., Wu, C. L., Illenye, S., Harlow, E., and Heintz, N. H. (1996). Nuclear
localization of DP and E2F transcription factors by heterodimeric partners and
retinoblastoma protein family members. J Cell Sci 109 ( Pt 7), 1717-1726.
Magnaghi-Jaulin, L., Groisman, R., Naguibneva, I., Robin, P., Lorain, S., Le Villain, J.
P., Troalen, F., Trouche, D., and Harel-Bellan, A. (1998). Retinoblastoma protein
represses transcription by recruiting a histone deacetylase. Nature 391, 601-605.
61
Maiti, B., Li, J., de Bruin, A., Gordon, F., Timmers, C., Opavsky, R., Patil, K., Tuttle, J.,
Cleghorn, W., and Leone, G. (2005). Cloning and characterization of mouse E2F8, a
novel mammalian E2F family member capable of blocking cellular proliferation. J Biol
Chem 280, 18211-18220.
Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J. F., Jr., Nelson, C. E., Kim, D. H.,
Kassel, J., Gryka, M. A., Bischoff, F. Z., Tainsky, M. A., and et al. (1990). Germ line p53
mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms.
Science 250, 1233-1238.
Malumbres, M., Sotillo, R., Santamaria, D., Galan, J., Cerezo, A., Ortega, S., Dubus, P.,
and Barbacid, M. (2004). Mammalian cells cycle without the D-type cyclin-dependent
kinases Cdk4 and Cdk6. Cell 118, 493-504.
Marchenko, N. D., Zaika, A., and Moll, U. M. (2000). Death signal-induced localization
of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem 275,
16202-16212.
Marin, M. C., Jost, C. A., Brooks, L. A., Irwin, M. S., O'Nions, J., Tidy, J. A., James, N.,
McGregor, J. M., Harwood, C. A., Yulug, I. G., et al. (2000). A common polymorphism
acts as an intragenic modifier of mutant p53 behaviour. Nat Genet 25, 47-54.
Marine, J. C., and Jochemsen, A. G. (2005). Mdmx as an essential regulator of p53
activity. Biochem Biophys Res Commun 331, 750-760.
Markey, M. P., Angus, S. P., Strobeck, M. W., Williams, S. L., Gunawardena, R. W.,
Aronow, B. J., and Knudsen, E. S. (2002). Unbiased analysis of RB-mediated
transcriptional repression identifies novel targets and distinctions from E2F action.
Cancer Res 62, 6587-6597.
Martelli, F., Hamilton, T., Silver, D. P., Sharpless, N. E., Bardeesy, N., Rokas, M.,
DePinho, R. A., Livingston, D. M., and Grossman, S. R. (2001). p19ARF targets certain
E2F species for degradation. Proc Natl Acad Sci U S A 98, 4455-4460.
Martinez-Balbas, M. A., Bauer, U. M., Nielsen, S. J., Brehm, A., and Kouzarides, T.
(2000). Regulation of E2F1 activity by acetylation. Embo J 19, 662-671.
Marzio, G., Wagener, C., Gutierrez, M. I., Cartwright, P., Helin, K., and Giacca, M.
(2000). E2F family members are differentially regulated by reversible acetylation. J Biol
Chem 275, 10887-10892.
Matsushime, H., Ewen, M. E., Strom, D. K., Kato, J. Y., Hanks, S. K., Roussel, M. F.,
and Sherr, C. J. (1992). Identification and properties of an atypical catalytic subunit
(p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell 71, 323-334.
Mayol, X., Grana, X., Baldi, A., Sang, N., Hu, Q., and Giordano, A. (1993). Cloning of a
new member of the retinoblastoma gene family (pRb2) which binds to the E1A
transforming domain. Oncogene 8, 2561-2566.
62
McCurrach, M. E., Connor, T. M., Knudson, C. M., Korsmeyer, S. J., and Lowe, S. W.
(1997). bax-deficiency promotes drug resistance and oncogenic transformation by
attenuating p53-dependent apoptosis. Proc Natl Acad Sci U S A 94, 2345-2349.
McKeller, R. N., Fowler, J. L., Cunningham, J. J., Warner, N., Smeyne, R. J., Zindy, F.,
and Skapek, S. X. (2002). The Arf tumor suppressor gene promotes hyaloid vascular
regression during mouse eye development. Proc Natl Acad Sci U S A 99, 3848-3853.
Melino, G., Lu, X., Gasco, M., Crook, T., and Knight, R. A. (2003). Functional
regulation of p73 and p63: development and cancer. Trends Biochem Sci 28, 663-670.
Meyer, C. A., Jacobs, H. W., Datar, S. A., Du, W., Edgar, B. A., and Lehner, C. F.
(2000). Drosophila Cdk4 is required for normal growth and is dispensable for cell cycle
progression. Embo J 19, 4533-4542.
Meyer, C. A., Jacobs, H. W., and Lehner, C. F. (2002). Cyclin D-cdk4 is not a master
regulator of cell multiplication in Drosophila embryos. Curr Biol 12, 661-666.
Meyerson, M., and Harlow, E. (1994). Identification of G1 kinase activity for cdk6, a
novel cyclin D partner. Mol Cell Biol 14, 2077-2086.
Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P., and Moll, U.
M. (2003). p53 has a direct apoptogenic role at the mitochondria. Mol Cell 11, 577-590.
Moberg, K., Starz, M. A., and Lees, J. A. (1996). E2F-4 switches from p130 to p107 and
pRB in response to cell cycle reentry. Mol Cell Biol 16, 1436-1449.
Moll, U. M., and Slade, N. (2004). p 6 3 and p73: roles in development and tumor
formation. Mol Cancer Res 2, 371-386.
Molofsky, A. V., He, S., Bydon, M., Morrison, S. J., and Pardal, R. (2005). Bmi-1
promotes neural stem cell self-renewal and neural development but not mouse growth
and survival by repressing the pl6Ink4a and pl9Arf senescence pathways. Genes Dev 19,
1432-1437.
Momand, J., Zambetti, G. P., Olson, D. C., George, D., and Levine, A. J. (1992). The
mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53mediated transactivation. Cell 69, 1237-1245.
Montes de Oca Luna, R., Wagner, D. S., and Lozano, G. (1995). Rescue of early
embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206.
Moore, L., Venkatachalam, S., Vogel, H., Watt, J. C., Wu, C. L., Steinman, H., Jones, S.
N., and Donehower, L. A. (2003). Cooperativity of p19ARF, Mdm2, and p53 in murine
tumorigenesis. Oncogene 22, 7831-7837.
63
Moroni, M. C., Hickman, E. S., Denchi, E. L., Caprara, G., Colli, E., Cecconi, F., Muller,
H., and Helin, K. (2001). Apaf-1 is a transcriptional target for E2F and p53. Nat Cell Biol
3, 552-558.
Mowat, M., Cheng, A., Kimura, N., Bernstein, A., and Benchimol, S. (1985).
Rearrangements of the cellular p53 gene in erythroleukaemic cells transformed by Friend
virus. Nature 314, 633-636.
Mudryj, M., Hiebert, S. W., and Nevins, J. R. (1990). A role for the adenovirus inducible
E2F transcription factor in a proliferation dependent signal transduction pathway. Embo J
9, 2179-2184.
Muller, H., Bracken, A. P., Vernell, R., Moroni, M. C., Christians, F., Grassilli, E.,
Prosperini, E., Vigo, E., Oliner, J. D., and Helin, K. (2001). E2Fs regulate the expression
of genes involved in differentiation, development, proliferation, and apoptosis. Genes
Dev 15, 267-285.
Murthy, S. C., Bhat, G. P., and Thimmappaya, B. (1985). Adenovirus ElIA early
promoter: transcriptional control elements and induction by the viral pre-early EIA gene,
which appears to be sequence independent. Proc Natl Acad Sci U S A 82, 2230-2234.
Nakano, K., and Vousden, K. H. (2001). PUMA, a novel proapoptotic gene, is induced
by p53. Mol Cell 7, 683-694.
Nakazawa, Y., Kamijo, T., Koike, K., and Noda, T. (2003). ARF tumor suppressor
induces mitochondria-dependent apoptosis by modulation of mitochondrial Bcl-2 family
proteins. J Biol Chem 278, 27888-27895.
Nicolas, E., Morales, V., Magnaghi-Jaulin, L., Harel-Bellan, A., Richard-Foy, H., and
Trouche, D. (2000). RbAp48 belongs to the histone deacetylase complex that associates
with the retinoblastoma protein. J Biol Chem 275, 9797-9804.
Niculescu, A. B., 3rd, Chen, X., Smeets, M., Hengst, L., Prives, C., and Reed, S. I.
(1998). Effects of p21(Cipl/Wafl) at both the G1/S and the G2/M cell cycle transitions:
pRb is a critical determinant in blocking DNA replication and in preventing
endoreduplication. Mol Cell Biol 18, 629-643.
O'Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V., and Mendell, J. T. (2005). c-
Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839-843.
Obert, S., O'Connor, R. J., Schmid, S., and Hearing, P. (1994). The adenovirus E4-6/7
protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F
complex. Mol Cell Biol 14, 1333-1346.
Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino, T.,
Taniguchi, T., and Tanaka, N. (2000). Noxa, a BH3-only member of the Bcl-2 family and
candidate mediator of p53-induced apoptosis. Science 288, 1053-1058.
64
Oeggerli, M., Tomovska, S., Schraml, P., Calvano-Forte, D., Schafroth, S., Simon, R.,
Gasser, T., Mihatsch, M. J., and Sauter, G. (2004). E2F3 amplification and
overexpression is associated with invasive tumor growth and rapid tumor cell
proliferation in urinary bladder cancer. Oncogene 23, 5616-5623.
Ogawa, H., Ishiguro, K., Gaubatz, S., Livingston, D. M., and Nakatani, Y. (2002). A
complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in GO
cells. Science 296, 1132-1136.
Ohtani, N., Zebedee, Z., Huot, T. J., Stinson, J. A., Sugimoto, M., Ohashi, Y., Sharrocks,
A. D., Peters, G., and Hara, E. (2001). Opposing effects of Ets and Id proteins on
pl6INK4a expression during cellular senescence. Nature 409, 1067-1070.
Ohtsubo, M., and Roberts, J. M. (1993). Cyclin-dependent regulation of G in
mammalian fibroblasts. Science 259, 1908-1912.
Okuda, M., Horn, H. F., Tarapore, P., Tokuyama, Y., Smulian, A. G., Chan, P. K.,
Knudsen, E. S., Hofmann, I. A., Snyder, J. D., Bove, K. E., and Fukasawa, K. (2000).
Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication. Cell 103,
127-140.
Oliner, J. D., Kinzler, K. W., Meltzer, P. S., George, D. L., and Vogelstein, B. (1992).
Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature
358, 80-83.
Oliner, J. D., Pietenpol, J. A., Thiagalingam, S., Gyuris, J., Kinzler, K. W., and
Vogelstein, B. (1993). Oncoprotein MDM2 conceals the activation domain of tumour
suppressor p53. Nature 362, 857-860.
Olive, K. P., Tuveson, D. A., Ruhe, Z. C., Yin, B., Willis, N. A., Bronson, R. T.,
Crowley, D., and Jacks, T. (2004). Mutant p53 gain of function in two mouse models of
Li-Fraumeni syndrome. Cell 119, 847-860.
Olivier, M., Hussain, S. P., Caron de Fromentel, C., Hainaut, P., and Harris, C. C. (2004).
TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of
cancer. IARC Sci Publ, 247-270.
Ormondroyd, E., de la Luna, S., and La Thangue, N. B. (1995). A new member of the DP
family, DP-3, with distinct protein products suggests a regulatory role for alternative
splicing in the cell cycle transcription factor DRTF1/E2F. Oncogene 11, 1437-1446.
Ortega, S., Malumbres, M., and Barbacid, M. (2002). Cyclin D-dependent kinases, INK4
inhibitors and cancer. Biochim Biophys Acta 1602, 73-87.
Ortega, S., Prieto, I., Odajima, J., Martin, A., Dubus, P., Sotillo, R., Barbero, J. L.,
Malumbres, M., and Barbacid, M. (2003). Cyclin-dependent kinase 2 is essential for
meiosis but not for mitotic cell division in mice. Nat Genet 35, 25-31.
65
Owen-Schaub, L. B., Angelo, L. S., Radinsky, R., Ware, C. F., Gesner, T. G., and Bartos,
D. P. (1995). Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis?
Cancer Lett 94, 1-8.
Page, B. D., Guedes, S., Waring, D., and Priess, J. R. (2001). The C. elegans E2F- and
DP-related proteins are required for embryonic asymmetry and negatively regulate
Ras/MAPK signaling. Mol Cell 7, 451-460.
Palmero, I., Murga, M., Zubiaga, A., and Serrano, M. (2002). Activation of ARF by
oncogenic stress in mouse fibroblasts is independent of E2F1 and E2F2. Oncogene 21,
2939-2947.
Palmero, I., Pantoja, C., and Serrano, M. (1998). p19ARF links the tumour suppressor
p53 to Ras. Nature 395, 125-126.
Pan, H., Yin, C., Dyson, N. J., Harlow, E., Yamasaki, L., and Van Dyke, T. (1998). Key
roles for E2F1 in signaling p53-dependent apoptosis and in cell division within
developing tumors. Mol Cell 2, 283-292.
Papathanasiou, M. A., Kerr, N. C., Robbins, J. H., McBride, O. W., Alamo, I., Jr.,
Barrett, S. F., Hickson, I. D., and Fornace, A. J., Jr. (1991). Induction by ionizing
radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase
C. Mol Cell Biol 11, 1009-1016.
Pardee, A. B. (1974). A restriction point for control of normal animal cell proliferation.
Proc Natl Acad Sci U S A 71, 1286-1290.
Parisi, T., Beck, A. R., Rougier, N., McNeil, T., Lucian, L., Werb, Z., and Amati, B.
(2003). Cyclins El and E2 are required for endoreplication in placental trophoblast giant
cells. Embo J 22, 4794-4803.
Park, I. K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L., Morrison, S.
J., and Clarke, M. F. (2003). Bmi-1 is required for maintenance of adult self-renewing
haematopoietic stem cells. Nature 423, 302-305.
Parrinello, S., Samper, E., Krtolica, A., Goldstein, J., Melov, S., and Campisi, J. (2003).
Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell
Biol 5, 741-747.
Pediconi, N., Ianari, A., Costanzo, A., Belloni, L., Gallo, R., Cimino, L., Porcellini, A.,
Screpanti, I., Balsano, C., Alesse, E., et al. (2003). Differential regulation of E2F1
apoptotic target genes in response to DNA damage. Nat Cell Biol 5, 552-558.
Petersen,
B. O., Wagener,
C., Marinoni,
F., Kramer, E. R., Melixetian,
M., Denchi, E. L.,
Gieffers, C., Matteucci, C., Peters, J. M., and Helin, K. (2000). Cell cycle- and cell
growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1. Genes
Dev 14, 2330-2343.
66
Pfeifer, G. P. (2000). p 5 3 mutational spectra and the role of methylated CpG sequences.
Mutat Res 450, 155-166.
Philpott, A., and Friend, S. H. (1994). E2F and its developmental regulation in Xenopus
laevis. Mol Cell Biol 14, 5000-5009.
Polager, S., Kalma, Y., Berkovich, E., and Ginsberg, D. (2002). E2Fs up-regulate
expression of genes involved in DNA replication, DNA repair and mitosis. Oncogene 21,
437-446.
Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin, L.,
Potes, J., Chen, K., Orlow, I., Lee, H. W., et al. (1998). The Ink4a tumor suppressor gene
product, pl9Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell
92, 713-723.
Powers, J. T., Hong, S., Mayhew, C. N., Rogers, P. M., Knudsen, E. S., and Johnson, D.
G. (2004). E2FI uses the ATM signaling pathway to induce p53 and Chk2
phosphorylation and apoptosis. Mol Cancer Res 2, 203-214.
Quelle, D. E., Ashmun, R. A., Shurtleff, S. A., Kato, J. Y., Bar-Sagi, D., Roussel, M. F.,
and Sherr, C. J. (1993). Overexpression of mouse D-type cyclins accelerates G1 phase in
rodent fibroblasts. Genes Dev 7, 1559-1571.
Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. (1995). Alternative reading
frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of
inducing cell cycle arrest. Cell 83, 993-1000.
Randle, D. H., Zindy, F., Sherr, C. J., and Roussel, M. F. (2001). Differential effects of
p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells
and macrophages. Proc Natl Acad Sci U S A 98, 9654-9659.
Rayman, J. B., Takahashi, Y., Indjeian, V. B., Dannenberg, J. H., Catchpole, S., Watson,
R. J., te Riele, H., and Dynlacht, B. D. (2002). E2F mediates cell cycle-dependent
transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor
complex. Genes Dev 16, 933-947.
Reilly, K. M., Loisel, D. A., Bronson, R. T., McLaughlin, M. E., and Jacks, T. (2000).
Nfl ;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects.
Nat Genet 26, 109-113.
Rempel, R. E., Saenz-Robles, M. T., Storms, R., Morham, S., Ishida, S., Engel, A., Jakoi,
L., Melhem, M. F., Pipas, J. M., Smith, C., and Nevins, J. R. (2000). Loss of E2F4
activity leads to abnormal development of multiple cellular lineages. Mol Cell 6, 293306.
Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A., and Dynlacht,
B. D. (2002). E2F integrates cell cycle progression with DNA repair, replication, and
G(2)/M checkpoints. Genes Dev 16, 245-256.
67
Ringrose, L., and Paro, R. (2004). Epigenetic regulation of cellular memory by the
Polycomb and Trithorax group proteins. Annu Rev Genet 38, 413-443.
Rodier, G., Makris, C., Coulombe, P., Scime, A., Nakayama, K., Nakayama, K. I., and
Meloche, S. (2005). p107 inhibits G1 to S phase progression by down-regulating
expression of the F-box protein Skp2. J Cell Biol 168, 55-66.
Rogers, K. T., Higgins, P. D., Milla, M. M., Phillips, R. S., and Horowitz, J. M. (1996).
DP-2, a heterodimeric partner of E2F: identification and characterization of DP-2
proteins expressed in vivo. Proc Natl Acad Sci U S A 93, 7594-7599.
Rogoff, H. A., Pickering, M. T., Debatis, M. E., Jones, S., and Kowalik, T. F. (2002).
E2F1 induces phosphorylation of p53 that is coincident with p53 accumulation and
apoptosis. Mol Cell Biol 22, 5308-5318.
Rogoff, H. A., Pickering, M. T., Frame, F. M., Debatis, M. E., Sanchez, Y., Jones, S., and
Kowalik, T. F. (2004). Apoptosis associated with deregulated E2F activity is dependent
on E2F1 and Atm/Nbsl/Chk2.
Mol Cell Biol 24, 2968-2977.
Rowland, B. D., Denissov, S. G., Douma, S., Stunnenberg, H. G., Bernards, R., and
Peeper, D. S. (2002). E2F transcriptional repressor complexes are critical downstream
targets of p19(ARF)/p53-induced proliferative arrest. Cancer Cell 2, 55-65.
Sage, J., Miller, A. L., Perez-Mancera, P. A., Wysocki, J. M., and Jacks, T. (2003). Acute
mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature 424,
223-228.
Sage, J., Mulligan, G. J., Attardi, L. D., Miller, A., Chen, S., Williams, B., Theodorou, E.,
and Jacks, T. (2000). Targeted disruption of the three Rb-related genes leads to loss of
G(1) control and immortalization. Genes Dev 14, 3037-3050.
Saito, S., Goodarzi, A. A., Higashimoto, Y., Noda, Y., Lees-Miller, S. P., Appella, E.,
and Anderson, C. W. (2002). ATM mediates phosphorylation at multiple p53 sites,
including Ser(46), in response to ionizing radiation. J Biol Chem 277, 12491-12494.
Sansome, C., Zaika, A., Marchenko, N. D., and Moll, U. M. (2001). Hypoxia death
stimulus induces translocation of p53 protein to mitochondria. Detection by
immunofluorescence on whole cells. FEBS Lett 488, 110-115.
Sarnow, P., Ho, Y. S., Williams, J., and Levine, A. J. (1982). Adenovirus Elb-58kd
tumor antigen and SV40 large tumor antigen are physically associated with the same 54
kd cellular protein in transformed cells. Cell 28, 387-394.
Sax, J. K., Fei, P., Murphy, M. E., Bernhard, E., Korsmeyer, S. J., and El-Deiry, W. S.
(2002). BID regulation by p53 contributes to chemosensitivity. Nat Cell Biol 4, 842-849.
68
Scheffner, M., Huibregtse, J. M., Vierstra, R. D., and Howley, P. M. (1993). The HPV-16
E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of
p53. Cell 75, 495-505.
Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J., and Howley, P. M.
(1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes
the degradation of p53. Cell 63, 1129-1136.
Scheijen, B., Bronk, M., van der Meer, T., and Bernards, R. (2003). Constitutive E2F1
overexpression delays endochondral bone formation by inhibiting chondrocyte
differentiation. Mol Cell Biol 23, 3656-3668.
Schlisio, S., Halperin, T., Vidal, M., and Nevins, J. R. (2002). Interaction of YY 1 with
E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function. Embo J
21, 5775-5786.
Sdek, P., Ying, H., Zheng, H., Margulis, A., Tang, X., Tian, K., and Xiao, Z. X. (2004).
The central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated
suppression of E2F and cell growth. J Biol Chem 279, 53317-53322.
Sears, R., Ohtani, K., and Nevins, J. R. (1997). Identification of positively and negatively
acting elements regulating expression of the E2F2 gene in response to cell growth
signals. Mol Cell Biol 17, 5227-5235.
Shan, B., Durfee, T., and Lee, W. H. (1996). Disruption of RB/E2F-1 interaction by
single point mutations in E2F-1 enhances S-phase entry and apoptosis. Proc Natl Acad
Sci U S A 93, 679-684.
Sharpless, N. E., Ramsey, M. R., Balasubramanian, P., Castrillon, D. H., and DePinho, R.
A. (2004). The differential impact of p16(INK4a) or pl9(ARF) deficiency on cell growth
and tumorigenesis. Oncogene 23, 379-385.
She, Q. B., Chen, N., and Dong, Z. (2000). ERKs and p38 kinase phosphorylate p53
protein at serine 15 in response to UV radiation. J Biol Chem 275, 20444-20449.
She, Q. B., Ma, W. Y., and Dong, Z. (2002). Role of MAP kinases in UVB-induced
phosphorylation of p53 at serine 20. Oncogene 21, 1580-1589.
Sheaff, R. J., Groudine, M., Gordon, M., Roberts, J. M., and Clurman, B. E. (1997).
Cyclin E-CDK2 is a regulator of p27Kipl. Genes Dev 11, 1464-1478.
Sherr, C. J. (1993). Mammalian Gi cyclins. Cell 73, 1059-1065.
Sherr, C. J. (2004). Principles of tumor suppression. Cell 116, 235-246.
Sherr, C. J., and DePinho, R. A. (2000). Cellular senescence: mitotic clock or culture
shock? Cell 102, 407-410.
69
Sherr, C. J., and Roberts, J. M. (1999). CDK inhibitors: positive and negative regulators
of Gi-phase progression. Genes Dev 13, 1501-1512.
Shieh, S. Y., Ikeda, M., Taya, Y., and Prives, C. (1997). DNA damage-induced
phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325-334.
Shieh, S. Y., Taya, Y., and Prives, C. (1999). DNA damage-inducible phosphorylation of
p53 at N-terminal sites including a novel site, Ser20, requires tetramerization. Embo J 18,
1815-1823.
Siliciano, J. D., Canman, C. E., Taya, Y., Sakaguchi, K., Appella, E., and Kastan, M. B.
(1997). DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev
11, 3471-3481.
Silva, R. L., Thornton, J. D., Martin, A. C., Rehg, J. E., Bertwistle, D., Zindy, F., and
Skapek, S. X. (2005). Arf-dependent regulation of Pdgf signaling in perivascular cells in
the developing mouse eye. Embo J 24, 2803-2814.
Simin, K., Wu, H., Lu, L., Pinkel, D., Albertson, D., Cardiff, R. D., and Van Dyke, T.
(2004). pRb inactivation in mammary cells reveals common mechanisms for tumor
initiation and progression in divergent epithelia. PLoS Biol 2, E22.
Sluss, H. K., Armata, H., Gallant, J., and Jones, S. N. (2004). Phosphorylation of serine
18 regulates distinct p53 functions in mice. Mol Cell Biol 24, 976-984.
Soengas, M. S., Alarcon, R. M., Yoshida, H., Giaccia, A. J., Hakem, R., Mak, T. W., and
Lowe, S. W. (1999). Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor
inhibition. Science 284, 156-159.
Stanelle, J., Stiewe, T., Theseling, C. C., Peter, M., and Putzer, B. M. (2002). Gene
expression changes in response to E2F1 activation. Nucleic Acids Res 30, 1859-1867.
Stevens, C., Smith, L., and La Thangue, N. B. (2003). Chk2 activates E2F-1 in response
to DNA damage. Nat Cell Biol 5, 401-409.
Stiewe, T., and Putzer, B. M. (2000). Role of the p53-homologue p73 in E2F1-induced
apoptosis. Nat Genet 26, 464-469.
Stommel, J. M., Marchenko, N. D., Jimenez, G. S., Moll, U. M., Hope, T. J., and Wahl,
G. M. (1999). A leucine-rich nuclear export signal in the p53 tetramerization domain:
regulation of subcellular localization and p53 activity by NES masking. Embo J 18,
1660-1672.
Storre, J., Elsasser, H. P., Fuchs, M., Ullmann, D., Livingston, D. M., and Gaubatz, S.
(2002). Homeotic transformations of the axial skeleton that accompany a targeted
deletion of E2f6. EMBO Rep 3, 695-700.
70
Strano, S., Munarriz, E., Rossi, M., Cristofanelli, B., Shaul, Y., Castagnoli, L., Levine, A.
J., Sacchi, A., Cesareni, G., Oren, M., and Blandino, G. (2000). Physical and functional
interaction between p53 mutants and different isoforms of p73. J Biol Chem 275, 2950329512.
Strobeck,
M. W., Knudsen, K. E., Fribourg,
A. F., DeCristofaro,
M. F., Weissman,
B. E.,
Imbalzano, A. N., and Knudsen, E. S. (2000). BRG-1 is required for RB-mediated cell
cycle arrest. Proc Natl Acad Sci U S A 97, 7748-7753.
Strobeck, M. W., Reisman, D. N., Gunawardena, R. W., Betz, B. L., Angus, S. P.,
Knudsen, K. E., Kowalik, T. F., Weissman, B. E., and Knudsen, E. S. (2002).
Compensation of BRG-1 function by Brm: insight into the role of the core SWI-SNF
subunits in retinoblastoma tumor suppressor signaling. J Biol Chem 277, 4782-4789.
Sugimoto, M., Kuo, M. L., Roussel, M. F., and Sherr, C. J. (2003). Nucleolar Arf tumor
suppressor inhibits ribosomal RNA processing. Mol Cell 11, 415-424.
Sugimoto, M., Martin, N., Wilks, D. P., Tamai, K., Huot, T. J., Pantoja, C., Okumura, K.,
Serrano, M., and Hara, E. (2002). Activation of cyclin D1-kinase in murine fibroblasts
lacking both p21(Cipl) and p27(Kipl). Oncogene 21, 8067-8074.
Suzuki, H., Kurita, M., Mizumoto, K., Nishimoto, I., Ogata, E., and Matsuoka, M.
(2003). pl9ARF-induced p53-independent apoptosis largely occurs through BAX.
Biochem Biophys Res Commun 312, 1273-1277.
Takahashi, Y., Rayman, J. B., and Dynlacht, B. D. (2000). Analysis of promoter binding
by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and
repression. Genes Dev 14, 804-816.
Talis, A. L., Huibregtse, J. M., and Howley, P. M. (1998). The role of E6AP in the
regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPVnegative cells. J Biol Chem 273, 6439-6445.
Tao, W., and Levine, A. J. (1999). P19(ARF) stabilizes p53 by blocking nucleo-
cytoplasmic shuttling of Mdm2. Proc Natl Acad Sci U S A 96, 6937-6941.
Taubert, S., Gorrini, C., Frank, S. R., Parisi, T., Fuchs, M., Chan, H. M., Livingston, D.
M., and Amati, B. (2004). E2F-dependent histone acetylation and recruitment of the
Tip60 acetyltransferase complex to chromatin in late G1. Mol Cell Biol 24, 4546-4556.
Taylor, W. R., and Stark, G. R. (2001). Regulation of the G2/M transition by p53.
Oncogene 20, 1803-1815.
Tibbetts, R. S., Brumbaugh, K. M., Williams, J. M., Sarkaria, J. N., Cliby, W. A., Shieh,
S. Y., Taya, Y., Prives, C., and Abraham, R. T. (1999). A role for ATR in the DNA
damage-induced phosphorylation of p53. Genes Dev 13, 152-157.
71
Tiemann, F., and Hinds, P. W. (1998). Induction of DNA synthesis and apoptosis by
regulated inactivation of a temperature-sensitive retinoblastoma protein. Embo J 17,
1040-1052.
Tolbert, D., Lu, X., Yin, C., Tantama, M., and Van Dyke, T. (2002). p19(ARF) is
dispensable for oncogenic stress-induced p53-mediated apoptosis and tumor suppression
in vivo. Mol Cell Biol 22, 370-377.
Trimarchi, J. M., Fairchild, B., Verona, R., Moberg, K., Andon, N., and Lees, J. A.
(1998). E2F-6, a member of the E2F family that can behave as a transcriptional repressor.
Proc Natl Acad Sci U S A 95, 2850-2855.
Trimarchi, J. M., Fairchild, B., Wen, J., and Lees, J. A. (2001). The E2F6 transcription
factor is a component of the mammalian Bmil-containing polycomb complex. Proc Natl
Acad Sci U S A 98, 1519-1524.
Trimarchi, J. M., and Lees, J. A. (2002). Sibling rivalry in the E2F family. Nat Rev Mol
Cell Biol 3, 11-20.
Trouche, D., Cook, A., and Kouzarides, T. (1996). The CBP co-activator stimulates
E2F1/DP1 activity. Nucleic Acids Res 24, 4139-4145.
Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L., and Jacks, T. (1998).
Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends
survival of Rb-deficient mouse embryos. Mol Cell 2, 293-304.
Tsai, K. Y., MacPherson, D., Rubinson, D. A., Crowley, D., and Jacks, T. (2002a). ARF
is not required for apoptosis in Rb mutant mouse embryos. Curr Biol 12, 159-163.
Tsai, K. Y., MacPherson, D., Rubinson, D. A., Nikitin, A. Y., Bronson, R., Mercer, K. L.,
Crowley, D., and Jacks, T. (2002b). ARF mutation accelerates pituitary tumor
development in Rb+/- mice. Proc Natl Acad Sci U S A 99, 16865-16870.
Unger, T., Juven-Gershon, T., Moallem, E., Berger, M., Vogt Sionov, R., Lozano, G.,
Oren, M., and Haupt, Y. (1999). Critical role for Ser20 of human p53 in the negative
regulation of p53 by Mdm2. Embo J 18, 1805-1814.
Unger, T., Mietz, J. A., Scheffner, M., Yee, C. L., and Howley, P. M. (1993). Functional
domains of wild-type and mutant p53 proteins involved in transcriptional regulation,
transdominant inhibition, and transformation suppression. Mol Cell Biol 13, 5186-5194.
Veltman, J. A., Fridlyand, J., Pejavar, S., Olshen, A. B., Korkola, J. E., DeVries, S.,
Carroll, P., Kuo, W. L., Pinkel, D., Albertson, D., et al. (2003). Array-based comparative
genomic hybridization for genome-wide screening of DNA copy number in bladder
tumors. Cancer Res 63, 2872-2880.
Vernell, R., Helin, K., and Muller, H. (2003). Identification of target genes of the
pl6INK4A-pRB-E2F pathway. J Biol Chem 278, 46124-46137.
72
Verona, R., Moberg, K., Estes, S., Starz, M., Vernon, J. P., and Lees, J. A. (1997). E2F
activity is regulated by cell cycle-dependent changes in subcellular localization. Mol Cell
Biol 17, 7268-7282.
Vogelstein, B., and Kinzler, K. W. (1993). The multistep nature of cancer. Trends Genet
9, 138-141.
Vogelstein, B., and Kinzler, K. W. (2004). Cancer genes and the pathways they control.
Nat Med 10, 789-799.
Wang, B., Liu, K., Lin, F. T., and Lin, W. C. (2004a). A role for 14-3-3 tau in E2F1
stabilization and DNA damage-induced apoptosis. J Biol Chem 279, 54140-54152.
Wang, Q., Hirohashi, Y., Furuuchi, K., Zhao, H., Liu, Q., Zhang, H., Murali, R., Berezov,
A., Du, X., Li, B., and Greene, M. I. (2004b). The centrosome in normal and transformed
cells. DNA Cell Biol 23, 475-489.
Weber, J. D., Jeffers, J. R., Rehg, J. E., Randle, D. H., Lozano, G., Roussel, M. F., Sherr,
C. J., and Zambetti, G. P. (2000). p53-independent functions of the p19(ARF) tumor
suppressor. Genes Dev 14, 2358-2365.
Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J., and Bar-Sagi, D. (1999).
Nucleolar Arf sequesters Mdm2 and activates p53. Nat Cell Biol 1, 20-26.
Wells, J., Graveel, C. R., Bartley, S. M., Madore, S. J., and Farnham, P. J. (2002). The
identification of E2F1-specific target genes. Proc Natl Acad Sci U S A 99, 3890-3895.
Wetmore, C., Eberhart, D. E., and Curran, T. (2001). Loss of p53 but not ARF
accelerates medulloblastoma in mice heterozygous for patched. Cancer Res 61, 513-516.
Williams, B. O., Remington, L., Albert, D. M., Mukai, S., Bronson, R. T., and Jacks, T.
(1994a). Cooperative tumorigenic effects of germline mutations in Rb and p53. Nat
Genet 7, 480-484.
Williams, B. O., Schmitt, E. M., Remington, L., Bronson, R. T., Albert, D. M., Weinberg,
R. A., and Jacks, T. (1994b). Extensive contribution of Rb-deficient cells to adult
chimeric mice with limited histopathological consequences. Embo J 13, 4251-4259.
Wirth, K. G., Ricci, R., Gimenez-Abian, J. F., Taghybeeglu, S., Kudo, N. R., Jochum,
W., Vasseur-Cognet, M., and Nasmyth, K. (2004). Loss of the anaphase-promoting
complex in quiescent cells causes unscheduled hepatocyte proliferation. Genes Dev 18,
88-98.
Woo, M. S., Sanchez, I., and Dynlacht, B. D. (1997). p1 3 0 and p107 use a conserved
domain to inhibit cellular cyclin-dependent kinase activity. Mol Cell Biol 17, 3566-3579.
Wu, C. L., Zukerberg, L. R., Ngwu, C., Harlow, E., and Lees, J. A. (1995). In vivo
association of E2F and DP family proteins. Mol Cell Biol 15, 2536-2546.
73
Wu, G. S., Burns, T. F., McDonald, E. R., 3rd, Jiang, W., Meng, R., Krantz, I. D., Kao,
G., Gan, D. D., Zhou, J. Y., Muschel, R., et al. (1997). KILLER/DR5 is a DNA damage-
inducible p53-regulated death receptor gene. Nat Genet 17, 141-143.
Wu, L., Timmers, C., Maiti, B., Saavedra, H. I., Sang, L., Chong, G. T., Nuckolls, F.,
Giangrande, P., Wright, F. A., Field, S. J., et al. (2001a). The E2F1-3 transcription
factors are essential for cellular proliferation. Nature 414, 457-462.
Wu, X., Bayle, J. H., Olson, D., and Levine, A. J. (1993). The p53-mdm-2 autoregulatory
feedback loop. Genes Dev 7, 1126-1132.
Wu, X., Webster, S. R., and Chen, J. (2001b). Characterization of tumor-associated Chk2
mutations. J Biol Chem 276, 2971-2974.
Wu, Z., Earle, J., Saito, S., Anderson, C. W., Appella, E., and Xu, Y. (2002). Mutation of
mouse p53 Ser23 and the response to DNA damage. Mol Cell Biol 22, 2441-2449.
Wunderlich, M., Ghosh, M., Weghorst, K., and Berberich, S. J. (2004). MdmX represses
E2F1 transactivation. Cell Cycle 3, 472-478.
Xiao, A., Wu, H., Pandolfi, P. P., Louis, D. N., and Van Dyke, T. (2002). Astrocyte
inactivation of the pRb pathway predisposes mice to malignant astrocytoma development
that is accelerated by PTEN mutation. Cancer Cell 1, 157-168.
Xu, H. J., Zhou, Y., Ji, W., Perng, G. S., Kruzelock, R., Kong, C. T., Bast, R. C., Mills,
G. B., Li, J., and Hu, S. X. (1997). Reexpression of the retinoblastoma protein in tumor
cells induces senescence and telomerase inhibition. Oncogene 15, 2589-2596.
Xu, M., Sheppard, K. A., Peng, C. Y., Yee, A. S., and Piwnica-Worms, H. (1994). Cyclin
A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1
by phosphorylation. Mol Cell Biol 14, 8420-8431.
Yamasaki, L., Bronson, R., Williams, B. O., Dyson, N. J., Harlow, E., and Jacks, T.
(1998). Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rbl(+/-)mice.
Nat Genet 18, 360-364.
Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996).
Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85, 537-548.
Yi, Y., Shepard, A., Kittrell, F., Mulac-Jericevic, B., Medina, D., and Said, T. K. (2004).
pl9ARF determines the balance between normal cell proliferation rate and apoptosis
during mammary gland development. Mol Biol Cell 15, 2302-2311.
Yin, C., Knudson, C. M., Korsmeyer, S. J., and Van Dyke, T. (1997). Bax suppresses
tumorigenesis and stimulates apoptosis in vivo. Nature 385, 637-640.
74
Young, A. P., and Longmore, G. D. (2004). Differences in stability of repressor
complexes at promoters underlie distinct roles for Rb family members. Oncogene 23,
814-823.
Yu, J., Wang, Z., Kinzler, K. W., Vogelstein, B., and Zhang, L. (2003). PUMA mediates
the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci U S A 100,
1931-1936.
Zajchowski, D. A., Boeuf, H., and Kedinger, C. (1985). The adenovirus-2 early EIIa
transcription unit possesses two overlapping promoters with different sequence
requirements for EIa-dependent stimulation. Embo J 4, 1293-1300.
Zetterberg, A., and Larsson, 0. (1985). Kinetic analysis of regulatory events in Gi
leading to proliferation or quiescence of Swiss 3T3 cells. Proc Natl Acad Sci U S A 82,
5365-5369.
Zhang, Y., and Chellappan, S. P. (1995). Cloning and characterization of human DP2, a
novel dimerization partner of E2F. Oncogene 10, 2085-2093.
Zhang, Y., Xiong, Y., and Yarbrough, W. G. (1998). ARF promotes MDM2 degradation
and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor
suppression pathways. Cell 92, 725-734.
Zhao, K., Chai, X., Johnston, K., Clements, A., and Marmorstein, R. (2001). Crystal
structure of the mouse p53 core DNA-binding domain at 2.7 A resolution. J Biol Chem
276, 12120-12127.
Zhao, Y., Chaiswing, L., Velez, J. M., Batinic-Haberle, I., Colburn, N. H., Oberley, T.
D., and St Clair, D. K. (2005). p 5 3 translocation to mitochondria precedes its nuclear
translocation and targets mitochondrial oxidative defense protein-manganese superoxide
dismutase. Cancer Res 65, 3745-3750.
Zheng, N., Fraenkel, E., Pabo, C. O., and Pavletich, N. P. (1999). Structural basis of
DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP. Genes Dev
13, 666-674.
Zhu, J., Jiang, J., Zhou, W., Zhu, K., and Chen, X. (1999). Differential regulation of
cellular target genes by p53 devoid of the PXXP motifs with impaired apoptotic activity.
Oncogene 18, 2149-2155.
Zhu, J., Zhang, S., Jiang, J., and Chen, X. (2000). Definition of the p53 functional
domains necessary for inducing apoptosis. J Biol Chem 275, 39927-39934.
Zhu, L., Harlow, E., and Dynlacht, B. D. (1995). p107 uses a p21CIP1-related domain to
bind cyclin/cdk2 and regulate interactions with E2F. Genes Dev 9, 1740-1752.
75
Ziebold, U., Lee, E. Y., Bronson, R. T., and Lees, J. A. (2003). E2F3 loss has opposing
effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but
metastasis of medullary thyroid carcinomas. Mol Cell Biol 23, 6542-6552.
Ziebold, U., Reza, T., Caron, A., and Lees, J. A. (2001). E2F3 contributes both to the
inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev
15, 386-391.
Zindy, F., Eischen, C. M., Randle, D. H., Kamijo, T., Cleveland, J. L., Sherr, C. J., and
Roussel, M. F. (1998). Myc signaling via the ARF tumor suppressor regulates p53dependent apoptosis and immortalization. Genes Dev 12, 2424-2433.
Zindy, F., Quelle, D. E., Roussel, M. F., and Sherr, C. J. (1997). Expression of the
pl6INK4a tumor suppressor versus other INK4 family members during mouse
development and aging. Oncogene 15, 203-211.
Zindy, F., Williams, R. T., Baudino, T. A., Rehg, J. E., Skapek, S. X., Cleveland, J. L.,
Roussel, M. F., and Sherr, C. J. (2003). Arf tumor suppressor promoter monitors latent
oncogenic signals in vivo. Proc Natl Acad Sci U S A 100, 15930-15935.
76
CHAPTER 2
Repression of the Arf tumor suppressor by E2F3
is required for normal cell cycle kinetics
Aaron Aslanian, Phillip J. Iaquinta, Raluca Verona and Jacqueline A. Lees
Author's contributions: Figure 1, 2, and 3A
77
ABSTRACT
Tumor development is dependent upon the inactivation of two key tumor
suppressor networks, p61Ink4 a-cycD/cdk4-pRB-E2Fand p19A-mdm2-p53, that regulate
cellular proliferation and the tumor surveillance response. These networks are known to
intersect with one another but the mechanisms are poorly understood. Here we show that
E2F directly participates in the transcriptional control of Arf in both normal and
transformed cells. This occurs in a manner that is significantly different from the
regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts
(MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In
quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function
was previously undetermined. E2f3-loss is sufficient to derepress Arf, triggering
activation of p53 and expression of p21C i p '. Thus, E2F3 is a key repressor of the pl9Af -
p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the
activation of p53 and p21Ci'p in E2f3-deficient MEFs. Arf-loss also rescues the known
cell cycle re-entry defect of E2f3 -'- cells and this correlates with restoration of appropriate
activation of classic E2F-responsive genes. Our data also demonstrates a direct role for
E2F in the oncogenic activation of Arf
Specifically, we observe recruitment of the
endogenous activating E2Fs, E2F1 and E2F3a, to the Arf promoter. Thus, distinct E2F
complexes directly contribute to the normal repression and oncogenic activation of Arf
We propose that monitoring of E2F levels and/or activity is a key component of Arf s
ability to respond to inappropriate, but not normal, cellular proliferation.
78
INTRODUCTION
The development of mammalian tumors is dependent upon the disruption of two
key biological activities, the control of cellular proliferation and the apoptotic response
(Hanahan and Weinberg, 2000). Remarkably, the Ink4a/Arf locus encodes two distinct
4
a and p19A f (pl4A in humans), that influence one or
tumor suppressor proteins, p16Ink
both of these processes (Chin et al., 1998; Sherr, 2001). p16Ink4 a is a core component of
the cell cycle control machinery (Sherr and Roberts, 1999). It controls the activity of the
G- kinase, cyclinDocdk4/6, and consequently the phosphorylation status of the pocket
protein family. This family includes the retinoblastoma protein (pRB) tumor suppressor
and its relatives, p107 and p130. In the unphosphorylated state, the pocket proteins bind
to the E2F family of transcription factors and prevent the expression of genes that are
essential for entry into, and passage through, the cell cycle (Trimarchi and Lees, 2002).
This inhibition occurs through two distinct mechanisms.
pRB binds to the activating
E2Fs, E2F1, 2 and 3a, and blocks their transcriptional activity. At the same time, the
repressive E2Fs, E2F4 and 5, recruit p107 or p130 and their associated histone
deactylases to E2F-responsive promoters. Under these conditions, the cell is blocked in
Go,/G. Mitogenic signaling activates cell cycle re-entry by allowing cyclinD-cdk4/6 to
overcome the repression by p16nk4a. The consequent phosphorylation of the pocket
proteins causes them to dissociate from E2F enabling activation of E2F-responsive genes.
In normal cells, the pl6Ink4 a-cyclinD-cdk4/6-pRB-E2F pathway responds to both positive
and negative growth regulatory signals to determine whether or not a cell will divide
(Sherr and Roberts, 1999). This pathway is disrupted in most, if not all, mammalian
I 4
tumors through loss of p16nk
a, up-regulation of cyclinD-cdk4/6 or loss of pRB (Sherr,
79
1996). The resulting deregulated proliferation is due, at least in part, to the inappropriate
activation of E2F (McCaffrey et al., 1999; Pan et al., 1998; Tsai et al., 1998; Yamasaki et
al., 1998; Ziebold et al., 2003; Ziebold et al., 2001).
The second product of the Ink4a/Arf locus, p19Arf,is a key component of the p53
tumor surveillance network (Sherr, 2001). p19Af exists at low or undetectable levels in
most normal cell and tissue types (Zindy et al., 2003). However, its expression is
specifically activated by abnormal proliferative signals. These include the continued in
vitro culturing of mouse embryonic fibroblasts (Kamijo et al., 1997) and the
inappropriate expression of proliferative oncogenes including activated ras, c-myc, E2F,
E1A and v-Abl (de Stanchina et al., 1998; Dimri et al., 2000; Palmero et al., 1998; Radfar
et al., 1998; Serrano et al., 1997; Zindy et al., 1998). Once it is expressed, p1 9 A f inhibits
the p53 ubiquitin ligase, mdm2, allowing activation of the p53 tumor suppressor (Honda
and Yasuda, 1999; Llanos et al., 2001; Pomerantz et al., 1998; Stott et al., 1998; Weber et
al., 1999; Zhang et al., 1998). Depending on the cellular context, p53 triggers either cell
cycle arrest (via induction of the cdk inhibitor, p21C iPl) or apoptosis (through activation of
various apoptosis inducers). In either case, this counteracts the effect of the abnormal
proliferative signals. Essentially, p19A acts as a defense to oncogenic signals. The
recent analysis of a mouse strain that expresses GFP in place of p19A, confirms that Arf
is induced by the oncogenic signals present in incipient tumors (Zindy et al., 2003). This
explains why inactivation of the p19Ar-p53 network is essential for the survival and
proliferation of tumor cells in vivo (Sherr, 2001).
The ability of Arf to specifically respond to inappropriate, but not normal,
proliferative signals must require a careful balance of transcriptional signals.
80
Understanding how this is achieved remains a major challenge. Numerous studies have
implicated E2F in this process (Phillips and Vousden, 2001). The Arfpromoter contains
consensus E2F binding sites and the over-expression of E2F1 is sufficient to trigger its
transcriptional activation (Bates et al., 1998; DeGregori et al., 1997). However, it is
unclear whether this regulation is direct because the identified E2F sites are not required
for E2F-dependent activation (Parisi et al., 2002) (Berkovich et al., 2003). There is also
considerable debate as to which E2F family members might activate Arf(Trimarchi and
Lees, 2002). Some groups conclude that this is an E2F1-specific activity while others
propose that this is a shared property of the activating E2Fs. Certainly, E2F1 is not
required for Arf induction in numerous settings (Baudino et al., 2003; Palmero et al.,
2002) and p19A" itself is dispensable for E2F-dependent apoptosis (Russell et al., 2002;
Tolbert et al., 2002; Tsai et al., 2002). These findings could reflect redundancy: perhaps
multiple E2Fs can activate a large panel of apoptotic inducers that includes p19A .
Alternatively, E2F may not contribute to Arf activation in vivo. Others have suggested
that Arfis regulated by repressive E2Fopocket protein complexes (Rowland et al., 2002).
However, unlike classic E2F-responsive genes, Arf is not appreciably induced during cell
cycle entry. Thus, if Arfis a genuine E2F target, it must be regulated in a distinct manner
from classic E2F-responsive genes. In this study, we use E2f3-deficient MEFs to probe
the role of E2F in Arf regulation. This analysis shows that a single member of the E2F
A under normal
family, E2f3, is required to maintain the transcriptional repression of p19'
proliferative conditions.
81
RESULTS
E2F3-loss causes induction of p19Arf and activation of p53 in primary cells.
Two E2F family members, E2F3a and E2F3b, are encoded by a single locus through the
use of different promoters and 5' coding exons (Leone et al., 2000). These proteins share
domains required for DNA binding, heterodimerization and pocket protein binding but
have distinct N-termini comprising either 122 (E2F3a) or 6 (E2F3b) amino acids. We
have previously generated an E2f3 mutant mouse strain that inactivates both of these
proteins. For simplicity, we refer to these mice as E2f3-'- or E2F3-deficient.
MEFs
derived from E2f3 -- animals typically have a reduced rate of proliferation (Humbert et al.,
2000). They also display a major defect in mitogen-induced cell cycle re-entry and a
corresponding impairment in the activation of all known E2F-responsive genes examined
(Humbert et al., 2000) (Figure 1A, B). Given these observations, we hypothesized that
p19A r f expression might be altered in E2F3-deficient MEFs if it is a bona fide E2F-target
gene. To test this notion, we compared the levels of p19A in early passage wild-type
versus E2f3-'- MEFs during cell cycle re-entry (Figure 1). Consistent with previous
studies (Sherr and DePinho, 2000), p19Af was barely detectable in the wild-type cells and
its expression did not vary significantly during the cell cycle (Figure IB). Strikingly,
while the expression of the classic E2F-responsive targets, cyclin A and p107, was lower
in the E2f3 -'- MEFs than the wild-type controls, that of p19A~ was greatly increased
(Figure B). Thus, E2F3-loss affects p19A r expression but in an entirely distinct manner
from other E2F-responsive genes.
82
Figure 1.
A
E
--
---
dU,UU
C-
70,00
60,00
0
50,00
40,00
a)
o
30,00
c
20,00
10,00
0o
.C
0
CO
e"
5
10
15
20
Time (hours)
3=,
B
E2f3+/+
E2f3-
12 16 20 24 0
0
--
12 16 20 24
.m
sVp107
G n
-_w-go
-,ro
w
0
cyclin A
'~,,"Ianm_
,W
f-lfl
C
25
,,,_, .
,,
~ p1 9Arf
,* *,* _1
ne-*1'
-
E2f3 +
12 16 20 24 0
E2f3' /
12 16 20 24
.....
,*,
*r
" -..L.2 '-..-
V*l
p16Ink4A
p53 activity
--
"''" p21
cipi
Figure 1. E2f3-deficient MEFs have increased levels of p19ARF, leading to
activation of p53. (A) Wild-type MEFs (solid line) and E2f3 knockout MEFs
(dotted line) were synchronized by serum starvation and cell cycle re-entry was
monitored by [3 H]-thymidine incorporation. (B, C) Total protein extracts were
prepared at the indicated times after stimulation with 10% serum and subjected to (B,
C) western blotting for cyclin A, p107, p19ARF, p16INK4A or p21CIP or (C)
electrophoretic mobility shift assay for active p53.
The predominant function of p19A f is to activate p53 by inhibiting its negative
regulator, the ubiquitin ligase mdm2. We therefore used electrophoretic mobility shift
assays (EMSA) to determine whether the induction of p1 9 Af in the E2f3 -'- MEFs affected
p53 activity (Figure
C, also 2B). In wild-type MEFs, p53 DNA-binding activity
remained low throughout the cell cycle.
p53 activity was elevated in E2F3-deficient
MEFs, especially during quiescence. The p53 protein also showed characteristic
hallmarks of activation: there was an increase in the levels of p53 that was
phosphorylated on Serine 15 and a subtle increase in the total p53 protein levels (data not
shown). Thus, deregulation of pl9Af in E2f3-' - MEFs is accompanied by the activation of
p53. A known downstream target of p53, the cdk inhibitor p21Cip , was also expressed at
higher levels in the E2F3-deficient MEFs (Figure C), as was pl16nk4 a, the other protein
expressed from the Ink4a/Arf locus (Figure 1B). It seemed likely that the elevated levels
of p19A r, p53 and p21C ip l might contribute to the defective cell cycle entry in the E2F3-
deficient MEFs.
The up-regulation of p1 9Arfaccounts for the cell cycle re-entry defect of the E2f3-'MEFs.
To address the biological consequences of p19A e up-regulation, we intercrossed
E2f3 and Arf mutant mice to generate E2f3-/-;Arf'- double mutant (DKO) MEFs. We then
examined the ability of these cells to re-enter the cell cycle relative to wild-type, E2f3-'-,
and Arf' controls (Figure 2A). Consistent with previous reports (Kamijo et al., 1997),
the Arf/' MEFs initiated DNA synthesis more rapidly than the wild-type counterparts.
Significantly, the cell cycle kinetics of the E2f3-'-;Arf'-DKO MEFs were
84
Figure 2.
A
i0
120,00
a
100,00)0
i'9
DO
..E2f3 I DKO
0o
0
60,00'0
.
40,00
10
20,00
I
.
-Arf '
80,0010
E
wildtype
-
0
0
Ch
5
10
20
15
25
Time (hours)
B
wildtype
0
12
16
E2f3 -
20 24
0
12
16
Arf --
20 24
0
12 16
20
DKO
24
0
12
16 20
24
.
-MrIP
p53 activity
p21 Cip1
M, M
!*IV
.
~-'*
. '
: ... w
cyclin A
p107
C
E]
120,
100,
80,
60,
F-
40,
E
IH:
20,
0
5
10
15
20
Time (hours)
25
Figure 2. Elimination of p]9Arf rescues cell cycle re-entry and p53 activation defects
of E2f3-deficient MEFs. (A) Wild-type MEFs (solid black line), E2f3 knockout
MEFs (dotted black line), pl9Arf knockout MEFs (solid grey line), and E2f3;p19Arf
double knockout MEFs (dotted grey line) were synchronized by serum starvation and
cell cycle re-entry was monitored by [ 3 H]-thymidine incorporation.
(B) Total protein
extracts were prepared at the indicated times after serum stimulation and subjected to
western blotting for cyclin A, p107 and p21CIP or electrophoretic mobility shift assay
for active p53. (C) Wild-type MEFs (solid black line), E2f3 knockout MEFs (dotted
black line), p53 knockout MEFs (solid grey line), and E2f3;p53 double knockout
MEFs (dashed grey line) were analyzed exactly as described above (A).
indistinguishable from those of the Arf'- MEFs. Consistent with this rescue, Arf-mutation
also suppressed the defective mitogen-induced activation of classic E2F-responsive
targets, such as cyclin A and p107, that exists in the E2f3 -' - cells (Figure 2B). Thus,
p19 Af-lossSis sufficient to over-ride the cell cycle re-entry defect of the E2f3 -'- MEFs.
EMSA and western blotting experiments showed that Arf mutation also suppressed the
activation of p53 and the induction of p21 CiP in the E2f3- - MEFs (Figure 2B), confirming
that these events are dependent upon the up-regulation of p1 9A f . Importantly, p53
mutation suppressed the cell cycle re-entry defect of the E2f3 -'- MEFs in an analogous
manner to Arf mutation (Figure 2C). This correlated with suppression of the p21CiPl
induction and restoration of appropriate E2F-responsive gene activation (data not shown).
Taken together, our findings indicate that E2f3 is required to inhibit Arf expression and
this plays an important role in regulating normal cell cycle re-entry by preventing
inappropriate activation of p53.
E2F3 directly contributes to the transcriptional regulation of Arf in normal cells.
Although numerous studies implicate E2F in Arf control, it has not been shown
that any E2F family member is a direct transcriptional regulator of this gene. To address
this question, we first asked whether E2F3-loss affects Arfat the transcriptional level.
Using quantitative RT-PCR we examined the mRNA levels of both Arf and a well-
characterized E2F-responsive gene, p 10 7, in wild-type versus E2F3-deficient MEFs
(Figure 3A).
As in our previous studies (Humbert et al., 2000), the mitogen-induced
activation of the p107 transcript was greatly impaired in the E2f3 -' - MEFs. In contrast,
ArfmRNA
levels were significantly higher in E2f3 -'- MEFs than the wild-type controls
86
Figure 3.
p107
A
5
C
Arf
2.5
o0 2
n
0
.n 4
CA
U)
1
2
75
:r
Er
- E2f3+1+
. E2f34
0.5n
IVlI
0
5
0
15
10
20
5
0
25
B
LL
LU
o
LL
U
C O
LLI
LU
20
15
25
.0
+
+
CM
10
Time (hours)
Time (hours)
W
... .
a 1.5.
83
a)
C
....
.....
..........
..I......... ..
o
_ .
:
0
o
a
LU
N
N
LU
UJ3
co
Nc
LU LU
'
Q
0
.
u_ u_oco
u_
LU LU L
LN
LU
a .
VVT
E2f3-
Arf
p107
D
C
control
-
Ca
0
Co
C')
co
C')
N
L
AS SS
_
f
c3a
_;
_
I
E2F3A
E2F3A -
E2F3B
E2F3B-:r
_
a
-
a
D-_
.C
Ard
'C-rer
lllle
I1IBLI
+b
I
I
p107
control
Figure 3. E2F3 is a direct repressor of Arf. (A) Wild-type (solid lines) or E2f3 knockout
(dotted lines) MEFs were synchronized by serum-starvation and RNA was extracted at the
indicated times after stimulation. Quantitative real-time RT-PCR analysis of p107 or pl9Arf
mRNA is shown. (B) ChIP analysis of asynchronous wild-type (WT) or E2f3 knockout MEFs.
Sonicated, cross-linked chromatin was immunoprecipitated with the indicated antibodies, and
the purified DNA was analyzed by PCR with primers specific for the p107 or Arf promoters, or
a control sequence lacking E2F sites (1 kb upstream of the E2fl promoter). Input, 0.5% of
chromatin in IP reactions was analyzed by PCR. (C) Schematic of the E2F3a and E2F3b
proteins, and the antibodies used in ChIP analysis (Black box, DNA-binding domain; light grey
box, dimerization domain; dark grey box, transactivation domain). (D) Cell lysates of
asynchronous (AS) wild-type MEFs or wild-type MEFs incubated in 0.1% serum for 3 days
(SS) were subjected to western blotting for the two E2F3 isoforms. ChIP assays for the indicated
E2F and pocket protein family members were performed on the serum starved wild-type MEFs.
(Figure 3A). This up-regulation was most striking in the Go time point (Figure 3A),
A Ar protein (Figure 1B).
where we observed the greatest increase in the levels of the p19
Thus, E2F3-Ioss is acting, at least in part, through changes in the levels of Arf mRNA.
To determine whether E2F3 is directly involved in Arf regulation, we used
chromatin immunopreciptiation (ChIP) to examine binding of E2F family members to the
Arf and p107 promoters (Figure 3B). In asynchronously growing wild-type MEFs, we
detected no significant enrichment of any E2F at a control sequence lacking E2F sites,
one kilobase upstream of the E2F1 promoter. In contrast, several E2F family members
could be detected at the pl07 promoter including E2F1, 2, 3 and 4 (Figure 3B, data not
shown). This is entirely consistent with the previous characterization of archetypal
mammalian E2F-responsive genes (Takahashi et al., 2000). Remarkably, the spectrum of
E2F proteins at the Arf promoter differed completely from this norm: we observed only a
single E2F, E2F3 (Figure 3B). This specificity was not restricted to murine cells, since
E2F3 was also the sole E2F bound to the p14Af promoter in both primary (WI-38) and
transformed (T98G, BJ-T) human cell lines (data not shown). In E2f3 -' - MEFs, there was
no longer any enrichment of the Arf or p107 promoter sequences by the anti-E2F3
antibodies, confirming their specificity (Figure 3B). Strikingly, E2F3-deficiency caused
a slight increase in the level of E2F1 associated with the p107 promoter but there was no
evidence of E2F 1, or any other E2F family member, binding to the Arf promoter in these
cells (Figure 3B). Taken together, these data show that E2F3 is directly involved in the
regulation of Arf, and that loss of E2F3, without apparent recruitment of E2F1, is
sufficient to allow Arf activation.
Based on these findings, we conclude that E2F3
represses Arf expression in normal cells.
88
It is well established that the E2F proteins can contribute to the repression of E2Fresponsive genes through recruitment of the pocket proteins, pRB, p107 and p130, and
their associated histone deactylases (Trimarchi and Lees, 2002). The E2F3 proteins have
been reported to bind to pRB, but not p107 or p130, in vivo (He et al., 2000; Leone et al.,
2000; Moberg et al., 1996). Given this finding, we used ChIP assays to determine
whether one or more pocket proteins were recruited to the Arf promoter along with E2F3
(Figure 3B). Consistent with previous studies, p130, but not pRB, was readily detected at
the p107 promoter. In contrast, none of the pocket proteins were detected at the Arf
promoter (Figure 3B, and data not shown; see also Figure 3D). It is entirely possible that
pRB is present but cannot be detected due to limitations in the assay, the antibodies or the
structure of the pRB/E2F3 repressive complex. However, we consistently detect pRB at
differentiation-associated
promoters using the same ChIP conditions (Tina Yuan and
J.A.L., unpublished observations). This raises the possibility that E2F3 mediates Arf
repression in a pocket protein-independent manner.
The E2F3 locus encodes two proteins, E2F3a and E2F3b, which differ only in
their N-terminal sequences (Leone et al., 2000) (Figure 3C). E2F3a has been linked to
the transcriptional activation of E2F-responsive genes, but this does not rule out a role in
repression. The transcriptional properties of E2F3b have not been established. To
discern which species of E2F3 is responsible for regulation of Arf, we made use of two
antibodies raised against the E2F3 proteins. The sc-879x antibody recognizes an epitope
on the unique amino-terminus of E2F3a, and therefore does not cross-react with E2F3b.
In contrast, a carboxyl-terminal antibody (sc-878x) recognizes both E2F3a and E2F3b.
For clarity, we will refer to these antibodies as E2F3a-specific or anti-E2F3a+b.
89
By
detecting ectopically expressed E2F3a and E2F3b proteins, we have confirmed the
specificity of these two reagents (data not shown). These experiments also showed that
the E2F3a-specific antibody has a considerably lower activity in ChIP assays than antiE2F3a+b (data not shown). Despite its low avidity, the E2F3a-specific antibody yielded
a detectable ChIP signal at the p107 promoter (Figure 3B). Under the same conditions,
we did not see any evidence of E2F3a binding to Arf(Figure 3B); however, because of
the poor avidity of the E2F3a-specific antibody, we cannot rule out that E2F3a
contributes to the repression of Arf, yet falls beneath the detection limit of this reagent.
To address this in an alternative way, we took advantage of the differential expression
patterns of the two E2F3 species. E2F3a expression is cell cycle regulated, peaking
during the G,/S transition, while E2F3b expression is constant throughout the cell cycle.
Consequently, E2F3b is the only E2F3 isoform expressed during quiescence (Figure 3D).
Therefore, we performed ChIP from MEFs that had been arrested in GO/G, by serum
deprivation (Figure 3D). In this setting, the p107 promoter was specifically occupied by
E2F4 and p1 30, the key components of the archetypal repressive E2F/pocket protein
complex. At the same time, we still specifically detected E2F3 at the Arf promoter using
the anti-E2F3a+b antibody.This analysis strongly implicates E2F3b in the repression of
Arf in quiescent cells, yet does not exclude contribution of E2F3a, along with E2F3b, in
asynchronous cells.
90
The activating E2Fs are directly involved in activation of Arf in response to
oncogenic stress.
It is well documented that Arfis a key regulator of the mammalian tumor
surveillance response network. Through an as yet undetermined mechanism, the
inappropriate expression of numerous oncogenes induces the expression of p19Af. The
resulting p53 activation triggers either cell cycle arrest (via p21C iPl induction) or apoptosis
(through activation of pro-apoptotic genes), thereby circumventing the oncogenes' ability
to drive inappropriate proliferation.
Having established a direct role for E2F3 in
repression of Arf in unstressed cells, we sought to determine what role E2F might play
during the activation of Arf by oncogenic challenge.
E2F1 is a potent oncogene. Its over-expression activates both cellular
proliferation and also the induction of p19Arf and high levels of apoptosis (Bates et al.,
1998; DeGregori et al., 1997). This latter response greatly impedes investigation of the
underlying molecular mechanisms. To overcome this problem, we have used MEFs in
which exons 2 and 3 of the Ink4a/Arf locus are deleted (Ink4a/Arf'
pl 6nk4a
MEFs) and therefore
and p1.9A f expression is disrupted (Serrano et al., 1996) (Figure 4A). This
mutation does not affect the upstream regulatory region of Arf, which is approximately
13kb upstream of the deletion, allowing us to examine E2F binding at the Arfpromoter
rf
the absence of apoptosis or other secondary events that might result from pl9A
induction. We infected these Ink4a/Arf'- MEFs with either control or E2F1-expressing
retroviruses and used ChIP to assess E2F binding to either the Arfor p107 promoters
(Figure 4B). When Ink4a/Arf'
MEFs were infected with a control retrovirus,
91
in
Figure 4.
A
,_
2
la p16
1I
_
3
1 2kb
,l
I
__
_
·
__
I_
_
C
_
p19Arf
_
Ink4aA
B
| °
0
vecTor
-
L
UIII
LL
N
III
LY
e=U
E2F1
EA
Ip107
__~~~~~~~
sit.es
E2F
Af
_
n3
C3
LL
N
III
ur
II
Od
U-
N
III
LY
cO
'I-
Y
o.
Il
__
C:
,
Co
I
0
,-
CM
CM
LL
U-
U-
w
W
W
LL
0r)
.
vector
E2F1
I~~~~~~~~~~~~~,,--II~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
E1A
Icontrol
|° 'E
(03
c
UL
r\l
LL
mI
C
c0
co
LL
C\M
'T
em
0
C
0a
vector
E2F1
E1A
Figure 4. The activating E2Fs bind to the Arf promoter during oncogenic challenge.
(A) Schematic of the mouse Ink4a/Arf locus, indicating the exon structure, and the
region deleted in the Ink4a/Arf knockout MEFs (bracket). Shaded regions in the
exons indicate regions coding for the pl6Ink4a (light grey) and p19Arf (black)
proteins. The small black boxes indicate two consensus E2F binding sites in the Arf
promoter, and the two small arrows indicate the location of the primers used in ChIP
analysis. (B) Ink4a/Arf knockout MEFs were infected with retrovirus overexpressing E2F1, E1A, or an empty virus (vector) and subjected to ChIP assays with
the indicated antisera.
the spectrum of E2F complexes detected at the Arf and p107 promoters was similar to
that observed in wild type, uninfected cells. Over-expression of E2F1 increased the level
of E2F1 associated with the p107 promoter, and caused a coordinate decrease in levels of
bound E2F3 and p130 (Figure 4B). This is consistent with the E2FI's known ability to
promote cell cycle progression and activate expression of classic E2F-responsive genes
(Trimarchi and Lees, 2002). Strikingly, we also detected significant levels of E2F1 at the
Arf promoter, showing that E2F1 can directly contribute to the transcriptional activation
of Arf when it is inappropriately expressed (Figure 4B).
We wished to determine whether the endogenous E2F1 participates in the
induction of Arf by other oncogenes. To address this question, we examined the effect of
over-expressing the adenoviral oncoprotein, EIA (Figure 4B). This promotes
proliferation and tumorigenesis by sequestering the pocket proteins and relieving the
transcriptional inhibition of their associated E2Fs (Ben-Israel and Kleinberger, 2002). In
Ink4a/Arf'- MEFs expressing E1A, we observed a decrease in p130 binding to the p107
promoter, and an increase in E2F1 binding, as expected (Figure 4B). Notably, we also
detected E2F binding to the Arf promoter, showing that the endogenous E2Fl protein
contributes to the activation of Arf arising from E1A expression. In addition, we detected
a weak signal with the E2F3a-specific antibody, indicating that E2F3a was cooperating
with E2F1 in the transcriptional regulation of Arf. Given these findings, we conclude that
the endogenous activating E2Fs play a direct role in the oncogene-induced activation of
Arf and the tumor surveillance network.
93
DISCUSSION
Taken together, our data show that the E2F proteins play a direct role in the
transcriptional regulation of Arf. This firmly establishes Arf as a bona-fide E2Fresponsive gene. However, the nature of this regulation diverges considerably from that
of archetypal E2F-responsive targets, for example genes encoding key components of the
cell cycle control and DNA replication machinery (Figure 5). These classic E2F-
responsive genes are expressed in a cell-cycle dependent manner that is orchestrated by
the specific binding of the repressive E2F/pocket protein complexes (predominantly
E2F4/p130) during Go/G, or the activating E2Fs (E2F1, 2 and 3a) during late G and G1/S
phase. In contrast, our data show that Arf is regulated by a more restricted subset of E2F
complexes whose activity is somehow determined by the stress-status, rather than the cell
cycle staging, of the cell.
In normal cells, Arf is constitutively repressed and our data show that this
correlates with the promoter binding of E2F3, and not other E2F family members. Since
the E2F3a-specific antibody works poorly in ChIP assay, our inability to detect a signal
with this reagent does not exclude the possibility that E2F3a contributes to Arf regulation.
In contrast, the presence of a robust anti-E2F3a+b ChIP signal in quiescent cells that
express E2F3b and not E2F3a, clearly shows that E2F3b is involved. At least in MEFs,
the absence of E2F3 is sufficient to trigger the expression of Arf, without obligatory
recruitment of the activating E2Fs. Thus, E2F3b (possibly in parallel with E2F3a) is
required for the constitutive repression of Arf in normal cells. Based on its high
expression in GO/GI cells, E2F3b had been proposed to function as a transcriptional
94
Figure 5.
Repression
Activation
II
classic E2F4/5
E2F
targets
Co
p 1 9 Arf
I
GI
G 1/S
C -Il
,-
F*
F-111'
Figure 5. Arfis regulated by E2F in a distinct manner from classic E2Fresponsive genes.
repressor (Leone et al., 2000). Our data provide the first direct evidence for this
hypothesis.
Remarkably, our analysis of the double mutant MEFs shows that Arf-loss
completely suppresses the cell cycle entry defect of the E2f3 -'- MEFs. This correlates
with the loss of activation of the p53 pathway and also restoration of the normal cell
cycle dependent activation of classic E2F-responsive genes. This genetic rescue
experiment is entirely consistent with the notion that E2F3 is acting to reppress Arf, and
therefore p53, in a linear pathway. However, it is also possible that the loss of Arf (or
p53) confers a proliferative advantage on the cells that outweighs or overrides the
proliferative disadvantage that results from E2f3-loss. In this scenario, Arf and E2f3
could be acting in parallel pathways or, alternatively, E2f3 could exert its effects through
both Arfl/p53-dependentand Arfl/p53-independentmechanisms. We currently favor the
second alternative. Taken together, the presence of E2F3 at the Arfpromoter
and the
derepression of Arf in E2f3-'- MEFs strongly support the existence of an Arf-dependent
mechanism.
However, additional experiments indicate that the Arf-p53 pathway does not
fully account for the phenotypic consequences of E2F3-loss. First, while loss of Arf or
p53 completely rescues the cell cycle re-entry defect of the E2f3 -' - MEFs, it only partially
suppresses their asynchronous proliferation defect (A.A. and J.A.L., unpublished
observations). Second, loss of Arf or p53 has no detectable effect on the developmental
phenotypes and the resulting embryonic/neonatal
lethality of the E2f3 -' - mice (A.A. and
J.A.L., unpublished observations). Thus, E2f3 has at least one essential function that is
Arf/p53-independent.
This second function remains to be established.
We had
previously concluded that the cell cycle re-entry defect of the E2f3-' - MEFs reflected a
96
direct role for E2f3 in the activation of classic E2F-responsive genes. However, this
current study shows that the defect in gene activation in the E2f3 -' - MEFs is fully reversed
by the loss of Arf or p53. Based on the same logic outlined above, these results can be
explained in two ways. First, the defect in activation of classic E2F-responsive genes in
the E2f3-'- cells could be an indirect consequence of activation of the Arf-p53 pathway.
Alternatively, E2f3 may be required for the appropriate activation of classic E2Fresponsive genes but the consequent loss of this activation in E2F3-deficient cells is
outweighed or overridden by the increased induction of these targets that results from the
loss of Arf or p53. Given the extensive cross-talk between the pRB and p53 pathways,
both models are highly plausible. More subtle experiments will be required to distinguish
between these two possibilities.
Our analysis of the E2f3 mutant cells reinforces a growing body of evidence that
the p19 Af-p5 3 network is the key determinant of the proliferation status of cultured
primary fibroblasts (Sherr, 2001). Several other transcriptional regulators, such as Bmil,
TBX2, TBX3 and Twist, have been linked to the repression of Arf and the inhibition of
p53 signaling (Brummelkamp et al., 2002; Jacobs et al., 2000; Jacobs et al., 1999;
Lingbeek et al., 2002; Maestro et al., 1999). There is limited insight into the
mechanism(s) of action of these repressors. In vitro promoter mapping studies show that
TBX2 and TBX3 can bind to a variant T-site located within the Initiator sequence of the
human Arf promoter (Lingbeek et al., 2002). However, promoter binding has not been
demonstrated in vivo. Similarly, antibodies against Bmil have not worked in ChIP
assays. This is thought to be an issue of antibody accessibility, since Bmi 1 is a
component of the multi-protein polycomb complex (van Lohuizen, 1998). However, it is
97
unclear how the Bmi 1-polycomb complex is recruited to Arf, with respect to either the
target DNA sequence/chromatin structure or the identity of the component(s) that
mediate the DNA/chromatin binding. Our identification of E2F3 as an additional
repressor of Arf raises additional questions. First, what is the precise nature of the
repressive E2F3 complex?
Is it a unique function of E2F3b or does E2F3a contribute?
Does it involve a higher order, antibody inaccessible pRB complex or does it function in
a pocket protein-independent manner? Second, what directs E2F3, but not other E2F
complexes, to bind specifically to the Arf promoter in normal cells? Finally, what is the
relationship between E2F3 and the other known Arfrepressors?
Do they function
independently of one another or work cooperatively to ensure the repression of Arf?
Careful analysis of both the repressor complexes and the Arf promoter will be required to
unravel this complexity.
Our data show that E2F is also involved in the activation of Arf in response to
oncogenic signals. Although there is extensive literature suggesting a link between E2F
and Arf(Trimarchi
and Lees, 2002), this is the first study to demonstrate that the action of
these proteins is direct. During oncogenic activation, we see recruitment of the
endogenous E2F1 and, to a lesser extent, the other activating E2Fs, E2F2 (P.I. and J.A.L.,
unpublished observations) and E2F3a. It is important to note that we do not completely
lose the anti-E2F3a+b signal at the Arf promoter under conditions of oncogenic stress
(Figure 4B). We believe that this partially reflects differences in the level of oncogenic
activation within the population of infected cells; some have sufficient E2F activation to
disrupt the E2F3b repression while others do not. Indeed, the continued presence of p130
at the p107 promoter in ElA-infected cells, strongly suggests that some cells express
98
insufficient E1A to fully dissociate the pocket protein/E2F complexes. A second
possibility is that the sustained anti-E2F3a+b signal at the induced Arf promoter reflects
recruitment of transcriptionally active E2F3a. We suspect that this is the case, at least in
the ElA-expressing
cells, because we see a weak signal with the E2F3a-specific antibody
and an enhanced signal with the anti-E2F3a+b antibody. Finally, since there are at least
two E2F consensus binding sites in the Arf promoter, we cannot rule out the possibility
that E2F3b remains bound to the Arf promoter during the stress response, despite
recruitment of E2F1 or other activating E2Fs. In this scenario, the activating E2Fs must
somehow override or negate the repressive function of E2F3b to ensure Arfinduction.
Importantly, this study provides considerable insight into the crosstalk between
the two key tumor suppressor networks, pRB-E2F and p19Ar-p53. It is already well
established that p53's growth suppressive properties are at least partially dependent upon
the ability of its downstream target, p2 1 CiP, to inhibit phosphorylation of pRB and
thereby promote its ability to repress E2F (Sherr, 2001). In this study, we now show that
E2F also plays a direct role in regulation of the p19AIf-p5 3 pathway. Consistent with
previous hypotheses, the activating E2Fs directly contribute to the induction of Arfthat
occurs in response to oncogenic stress, frequently referred to as the tumor surveillance
response. In addition, our data show that E2F3 plays a key role in maintaining the p19A rp53 network in the repressed state when there is no oncogenic stress signal. There has
been considerable debate as to how a cell could know whether it initiates inappropriate,
as opposed to appropriate, proliferation and regulate Arf accordingly (Lowe and Sherr,
2003). It is well known that the state of E2F complexes is a key determinant of whether
normal cells will divide. Our current finding that various E2F complexes contribute to
99
both the repressed and activated states of Arf regulation strongly suggests that monitoring
of E2F levels and/or activity is likely to lie at the heart of this tumor surveillance
mechanism.
100
MATERIALS AND METHODS
Generation of mouse strains and MEF preparation
E2f3+/';Arf'- and E2f3+/-;p53+/- mice were generated by intercrossing E2f3 +'/ mice
(Humbert et al., 2000) with either Arf ' mice (Kamijo et al., 1997) or p53 +' (Williams et
al., 1994) respectively. Genotyping was performed as previously described. Double
heterozygous mutant mice were intercrossed and MEFs were prepared from e13.5
embryos as previously described (Humbert et al., 2000). Ink4a/Arf'- mice (Serrano et al.,
1996) were used to generate Ink4a/Arf'- MEFs.
Serum starvation and release experiments
Passage 4 MEFs were plated in triplicate onto 3.5-cm dishes at 2 x 105 cells/dish and cell
cycle re-entry was performed as previously described (Humbert et al., 2000). After 48
hours, cells were washed twice with DME and then incubated in low serum (0.1I% FCS)
for 72 hours. Cells were subsequently fed with media containing 10% FCS. For each
timepoint, cells were labeled with 5[FCi [ 3 H]-thymidine for 1 hour. Cells were harvested
and [ 3 H]-thymidine incorporation was measured as previously described (Moberg et al.,
1996).
RNA preparation and Quantitative Real-time PCR analysis
Passage 4 MEFs were plated onto 15-cm dishes at 3 x 106 cells/dish and cell cycle reentry was performed as described above. For each time point, cells were harvested and
total RNA was isolated using RNeasy (Qiagen) and an on-column DNAse step was
performed according to manufacturer's instructions. cDNAs were generated with
Superscript First Strand Synthesis System (Invitrogen). Quantitative realtime PCR
reactions using SYBR Green dye (Applied Biosystems) were run in triplicate on an ABI
Prism 7000 Real-time PCR machine (Applied Biosystems). Control 18S rRNA reactions
101
(Applied Biosystems) were also run to normalize ACt values. Fold change was
calculated as 2A(AACt). Primer sequences for Arf were 5'CACCGGAATCCTGGACCAG-3' and 5'-GCAGTTCGAATCTGCACCGT-3' and for
p107 were 5'-GATGCTCATCTGACCGGAGT-3'
and 5'-
ATAAGTCACGTAGGCGCACA-3'.
Protein preparation, western blotting and gel retardation assays
Passage 4 MEFs were plated onto 15-cm dishes at 3 x 106 cells/dish and cell cycle reentry was performed as described above. For each time point, cells were harvested and
total protein was isolated as described previously (Moberg et al., 1996). Western blotting
was performed using
OO0tgwhole cell extract using anti-cyclin A (Santa Cruz sc-596),
anti-p107 (Santa Cruz sc318), anti-pl9
A
(Novus NB200-106), anti-p16nk4 a (Santa Cruz
sc-1207), anti-p 2 1CiP (Santa Cruz sc-6246). In Figure 3D, extracts were prepared as
above from asynchronous MEFs, or MEFs incubated in 0.1I% serum for 3 days. Western
blotting was performed on 30tg extract using the anti-E2F3a+b antibody (sc-878, Santa
Cruz). p53 gel retardation assays were performed in the presence of supershifting
antibody using NuShift (Geneka) according to the manufacturer's instructions.
Retroviral Infection
Infections were performed exactly as described (Serrano et al., 1997), except that 15-cm
plates were used at all steps, and the procedure was scaled up accordingly. Target
Ink4a/Arf'- MEFs were selected for two days in 2 mg/mL puromycin, grown for a further
two days, and then subjected to ChIP analysis as described below. pBabe-Puro, pBabePuro-E2F1 (Humbert et al., 2000), and LPC-12S-E1A
Stanchina et al., 1998).
102
have been described (de
Chromatin Immunoprecipitation (ChIP)
ChIP was performed essentially as described (Takahashi et al., 2000). Early-passage
(passage 3-5) MEFs were used for all experiments. Sonicated, cross-linked chromatin
corresponding to approximately 3x106 cells was immunoprecipitated
with the following
antibodies: E2F1, sc-193; E2F3a, sc-879x; E2F3a+b, sc-878x; E2F4, sc-1082x; p130, sc317x (all from Santa Cruz); pRB, MS-594 and MS-595 (Neomarkers); control (antiLuciferase) 05-603 (Upstate Biotechnology). 3-4% of the precipitated DNA, or 0.5%
input DNA, was amplified by thirty cycles of PCR using the primer sequences: p107 (5'TTAGAGTCCGAGGTCCATCTTCT-3' and 5'-GGGCTCGTCCTCGAACATATCC3'); Arf (5'-GC1'GGCTGTCACCGCGAT-3' and 5'-GCGTTGAGGCACCTCGAGA3'); E2fl-upstream (control) (5'-TGGAGGTCAAGTAGTGGCCCAAA-3' and 5'ACAATGTCTGGTTTGCTCCGCCC-3').
PCR products were resolved on 8%
polyacrylamide gels and stained with ethidium bromide.
103
ACKNOWLEDGEMENTS
We thank Chuck Sherr, Tyler Jacks and Ron DePinho for providing Arf, p53 and
Ink4a/Arfmutant mice and also for stimulating discussions of this project. We are also
grateful to Phil Sharp, Steve Bell and members of the Lees lab for helpful discussions
during this study and the preparation of this manuscript. J.A.L. is a Daniel K. Ludwig
Scholar. This work was supported by a grant from the NIH (PO1-CA42063) to J.A.L.
104
References.
Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden,
K. H. (1998). p14ARF links the tumour suppressors RB and p53. Nature 395, 124-125.
Baudino, T. A., Maclean, K. H., Brennan, J., Parganas, E., Yang, C., Aslanian, A., Lees,
J. A., Sherr, C. J., Roussel, M. F., and Cleveland, J. L. (2003). Myc-mediated
proliferation and lymphomagenesis, but not apoptosis, are compromised by E2fl loss.
Mol Cell 11, 905-914.
Ben-Israel, H., and Kleinberger, T. (2002). Adenovirus and cell cycle control. Front
Biosci 7, d1369-1395.
Berkovich, E., Lamed, Y., and Ginsberg, D. (2003). E2F and Ras synergize in
transcriptionally activating p14ARF expression. Cell Cycle 2, 127-133.
Brummelkamp, T. R., Kortlever, R. M., Lingbeek, M., Trettel, F., MacDonald, M. E., van
Lohuizen, M., and Bernards, R. (2002). TBX-3, the gene mutated in Ulnar-Mammary
Syndrome, is a negative regulator of p19ARF and inhibits senescence. J Biol Chem 277,
6567-6572.
Chin, L., Pomerantz, J., and DePinho, R. A. (1998). The INK4a/ARF tumor suppressor:
one gene--two products--two pathways. Trends Biochem Sci 23, 291-296.
de Stanchina, E., McCurrach, M. E., Zindy, F., Shieh, S. Y., Ferbeyre, G., Samuelson, A.
V., Prives, C., Roussel, M. F., Sherr, C. J., and Lowe, S. W. (1998). E1A signaling to p53
involves the p19(ARF) tumor suppressor. Genes Dev 12, 2434-2442.
DeGregori, J., Leone, G., Miron, A., Jakoi, L., and Nevins, J. R. (1997). Distinct roles for
E2F proteins in cell growth control and apoptosis. Proc Natl Acad Sci U S A 94, 7245-
7250.
Dimri, G. P., Itahana, K., Acosta, M., and Campisi, J. (2000). Regulation of a senescence
checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor.
Mol Cell Biol 20, 273-285.
Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.
He, Y., Armanious, M. K., Thomas, M. J., and Cress, W. D. (2000). Identification of
E2F-3B, an alternative form of E2F-3 lacking a conserved N-terminal region. Oncogene
19, 3422-3433.
Honda, R., and Yasuda, H. (1999). Association of pl9(ARF) with Mdm2 inhibits
ubiquitin ligase activity of Mdm2 for tumor suppressor p53. Embo J 18, 22-27.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
105
Jacobs, J. J., Keblusek, P., Robanus-Maandag, E., Kristel, P., Lingbeek, M., Nederlof, P.
M., van Welsem, T., van de Vijver, M. J., Koh, E. Y., Daley, G. Q., and van Lohuizen,
M. (2000). Senescence bypass screen identifies TBX2, which represses Cdkn2a
(p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet 26, 291-299.
Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A., and van Lohuizen, M. (1999).
The oncogene and Polycomb-group gene bmi-l regulates cell proliferation and
senescence through the ink4a locus. Nature 397, 164-168.
Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing, J. R., Ashmun, R. A.,
Grosveld, G., and Sherr, C. J. (1997). Tumor suppression at the mouse INK4a locus
mediated by the alternative reading frame product p19ARF. Cell 91, 649-659.
Leone, G., Nuckolls, F., Ishida, S., Adams, M., Sears, R., Jakoi, L., Miron, A., and
Nevins, J. R. (2000). Identification of a novel E2F3 product suggests a mechanism for
determining specificity of repression by Rb proteins. Mol Cell Biol 20, 3626-3632.
Lingbeek, M. E., Jacobs, J. J., and van Lohuizen, M. (2002). The T-box repressors TBX2
and TBX3 specifically regulate the tumor suppressor gene p14ARF via a variant T-site in
the initiator. J Biol Chem 277, 26120-26127.
Llanos, S., Clark, P. A., Rowe, J., and Peters, G. (2001). Stabilization of p53 by p14ARF
without relocation of MDM2 to the nucleolus. Nat Cell Biol 3, 445-452.
Lowe, S. W., and Sherr, C. J. (2003). Tumor suppression by Ink4a-Arf: progress and
puzzles. Curr Opin Genet Dev 13, 77-83.
Maestro, R., Dei Tos, A. P., Hamamori, Y., Krasnokutsky, S., Sartorelli, V., Kedes, L.,
Doglioni, C., Beach, D. H., and Hannon, G. J. (1999). Twist is a potential oncogene that
inhibits apoptosis. Genes Dev 13, 2207-2217.
McCaffrey, J., Yamasaki, L., Dyson, N. J., Harlow, E., and Griep, A. E. (1999).
Disruption of retinoblastoma protein family function by human papillomavirus type 16
E7 oncoprotein inhibits lens development in part through E2F-1. Mol Cell Biol 19, 64586468.
Moberg, K., Starz, M. A., and Lees, J. A. (1996). E2F-4 switches from p130 to p107 and
pRB in response to cell cycle reentry. Mol Cell Biol 16, 1436-1449.
Palmero, I., Murga, M., Zubiaga, A., and Serrano, M. (2002). Activation of ARF by
oncogenic stress in mouse fibroblasts is independent of E2F1 and E2F2. Oncogene 21,
2939-2947.
Palmero, I., Pantoja, C., and Serrano, M. (1998). p19ARF links the tumour suppressor
p53 to Ras. Nature 395, 125-126.
106
Pan, H., Yin, C., Dyson, N. J., Harlow, E., Yamasaki, L., and Van Dyke, T. (1998). Key
roles for E2F1 in signaling p53-dependent apoptosis and in cell division within
developing tumors. Mol Cell 2, 283-292.
Parisi, T., Pollice, A., Di Cristofano, A., Calabro, V., and La Mantia, G. (2002).
Transcriptional regulation of the human tumor suppressor p14(ARF) by E2F1, E2F2,
E2F3, and Spl-like factors. Biochem Biophys Res Commun 291, 1138-1145.
Phillips, A. C., and Vousden, K. H. (2001). E2F-1 induced apoptosis. Apoptosis 6, 173182.
Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin, L.,
Potes, J., Chen, K., Orlow, I., Lee, H. W., et al. (1998). The Ink4a tumor suppressor gene
product, pl9Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell
92, 713-723.
Radfar, A., Unnikrishnan, I., Lee, H. W., DePinho, R. A., and Rosenberg, N. (1998).
p19(Arf) induces p53-dependent apoptosis during abelson virus-mediated pre-B cell
transformation. Proc Natl Acad Sci U S A 95, 13194-13199.
Rowland, B. D., Denissov, S. G., Douma, S., Stunnenberg, H. G., Bernards, R., and
Peeper, D. S. (2002). E2F transcriptional repressor complexes are critical downstream
targets of pl9(ARF)/p53-induced proliferative arrest. Cancer Cell 2, 55-65.
Russell, J. L., Powers, J. T., Rounbehler, R. J., Rogers, P. M., Conti, C. J., and Johnson,
D. G. (2002). ARF differentially modulates apoptosis induced by E2F1 and Myc. Mol
Cell Biol 22, 1360-1368.
Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D., and DePinho, R. A. (1996).
Role of the INK4a locus in tumor suppression and cell mortality. Cell 85, 27-37.
Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe, S. W. (1997).
Oncogenic ras provokes premature cell senescence associated with accumulation of p53
and pl6INK4a. Cell 88, 593-602.
Sherr, C. J. (1996). Cancer cell cycles. Science 274, 1672-1677.
Sherr, C. J. (2001). The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell
Biol 2, 731-737.
Sherr, C. J., and DePinho, R. A. (2000). Cellular senescence: mitotic clock or culture
shock? Cell 102, 407-410.
Sherr, C. J., and Roberts, J. M. (1999). CDK inhibitors: positive and negative regulators
of GI-phase progression. Genes Dev 13, 1501-1512.
Stott, F. J., Bates, S., James, M. C., McConnell, B. B., Starborg, M., Brookes, S.,
Palmero, I., Ryan, K., Hara, E., Vousden, K. H., and Peters, G. (1998). The alternative
107
product from the human CDKN2A locus, p14(ARF), participates in a regulatory
feedback loop with p53 and MDM2. Embo J 17, 5001-5014.
Takahashi, Y'., Rayman, J. B., and Dynlacht, B. D. (2000). Analysis of promoter binding
by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and
repression. Genes Dev 14, 804-816.
Tolbert, D., Lu, X., Yin, C., Tantama, M., and Van Dyke, T. (2002). pl9(ARF) is
dispensable for oncogenic stress-induced p53-mediated apoptosis and tumor suppression
in vivo. Mol Cell Biol 22, 370-377.
Trimarchi, J. M., and Lees, J. A. (2002). Sibling rivalry in the E2F family. Nature
Reviews Mol Cell Biol 3, 11-20.
Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L., and Jacks, T. (1998).
Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends
survival of Rb-deficient mouse embryos. Mol Cell 2, 293-304.
Tsai, K. Y., MacPherson, D., Rubinson, D. A., Crowley, D., and Jacks, T. (2002). ARF is
not required for apoptosis in Rb mutant mouse embryos. Curr Biol 12, 159-163.
van Lohuizen, M. (1998). Functional analysis of mouse Polycomb group genes. Cell Mol
Life Sci 54, 71-79.
Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J., and Bar-Sagi, D. (1999).
Nucleolar Arf sequesters Mdm2 and activates p53. Nat Cell Biol 1, 20-26.
Williams, B. O., Remington, L., Albert, D. M., Mukai, S., Bronson, R. T., and Jacks, T.
(1994). Cooperative tumorigenic effects of germline mutations in Rb and p53. Nat Genet
7, 480-484.
Yamasaki, L., Bronson, R., Williams, B. O., Dyson, N. J., Harlow, E., and Jacks, T.
(1998). Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rbl(+/-)mice.
Nat Genet 18, 360-364.
Zhang, Y., Xiong, Y., and Yarbrough, W. G. (1998). ARF promotes MDM2 degradation
and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor
suppression pathways. Cell 92, 725-734.
Ziebold, U., Lee, E. Y., Bronson, R. T., and Lees, J. A. (2003). E2F3 loss has opposing
effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but
metastasis of medullary thyroid carcinomas. Mol Cell Biol 23, 6542-6552.
Ziebold, U., Reza, T., Caron, A., and Lees, J. A. (2001). E2F3 contributes both to the
inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev
15, 386-391.
108
Zindy, F., Eischen, C. M., Randle, D. H., Kamijo, T., Cleveland, J. L., Sherr, C. J., and
Roussel, M. F. (1998). Myc signaling via the ARF tumor suppressor regulates p53-
dependent apoptosis and immortalization. Genes Dev 12, 2424-2433.
Zindy, F., Williams, R. T., Baudino, T. A., Rehg, J. E., Skapek, S. X., Cleveland, J. L.,
Roussel, M. F., and Sherr, C. J. (2003). Arf tumor suppressor promoter monitors latent
oncogenic signals in vivo. Proc Natl Acad Sci U S A 100, 15930-15935.
109
CHAPTER
3
The proliferation defect in cycling E2f3-deficient cells is pl9Arf-independent
Aaron Aslanian and Jacqueline A. Lees
Author's contributions: Figure 1, 2, 3, and 4
110
SUMMARY
The E2F transcription factor is a key regulator of cell cycle progression in both
normal and transformed cells (Attwooll et al., 2004; Blais and Dynlacht, 2004).
However, E2F also regulates a number of genes with functions beyond that of basic cell
cycle control (Muller et al., 2001; Ren et al., 2002). One such target is the tumor
suppressor protein p19ARF, which is an important positive regulator of the p53 pathway
during tumor surveillance (Lowe and Sherr, 2003). Induction of p19ARF inhibits cell
cycle progression (Quelle et al., 1995). During cell cycle re-entry, loss of E2f3 causes
de-repression ofpl9Arf, resulting in activation of p53, failure to properly induce classic
E2F targets, and delayed cell cycle re-entry kinetics. Deletion of pl9Arf completely
suppresses these defects (Aslanian et al., 2004). We sought to determine whether E2F3
regulates cellular proliferation in cycling cells similarly. Although asynchronously
growing E2f3-deficient cells possess a proliferation defect, there was no alteration in
p19ARF expression or in the expression of classic E2F targets examined in this study.
Furthermore, deletion of pl9Arf failed to suppress the proliferation defect. The loss of
E2f3 also impaired transformed cells as well as tumor development in a pl9Arfindependent manner. From these studies, we conclude that E2F3 regulates cellular
proliferation in cycling cells distinctly from its control of cell cycle re-entry, and in a
manner that is independent of its regulation of pl9Arf expression.
111
RESULTS AND DISCUSSION
Mouse embryonic fibroblasts (MEFs) have been used to examine the role of
numerous E2fs in cell cycle control. While the majority of the E2f knockouts (E2fl,
E2f4, E2f5 and E2f6) have no obvious proliferation defects, E2f3-deficient MEFs are
profoundly impaired (Gaubatz et al., 2000; Humbert et al., 2000a; Humbert et al., 2000b;
Rempel et al., 2000; Storre et al., 2002). They display defects in mitogen-induced cell
cycle re-entry and activation of classic E2F-responsive genes and they also have a
reduced rate of asynchronous proliferation (Humbert et al., 2000b). We have recently
shown that E2F3, and not other E2F proteins, contributes to the transcriptional repression
of the Arftumor suppressor in normal MEFs (Aslanian et al., 2004). p1 9A rf is elevated in
E2f3-deficient MEFs during cell cycle re-entry, resulting in hyper-activation of p53 and
induction of p21CIPl(Aslanian et al., 2004). Genetic analysis suggests that the derepression of the Arf-p53 pathway accounts for the defect in mitogen-induced cell cycle
re-entry and the E2F-responsive gene activation in the E2f3-deficient MEFs. Given this
observation, we wished to determine how the other consequences of E2f3-deficiency
relate to E2F3's role in Arf regulation.
The asynchronous proliferation defect of E2f3-deficient cells does not involve p19Af.
Our first goal was to examine E2f3's requirement in asynchronous proliferation
(Figure 1). Consistent with our previous studies, E2f3 -/' MEFs accumulate at a slower
rate than the wildtype controls but there is no evidence of increased apoptosis (Figure 1A;
data not shown). Since the E2F transcription factors play a key role in regulating S-phase
112
Figure 1.
A
B
16
C
2
Go
8
8
II
o
'Z54
c
c
8?
c'o
0.
E
1.2
1P
Ic
3
/wr E2f3
4
days
D
F
W
""of
p107
cyclinA
_/
p19ARF
-
*.
I
o
2'Vl
F
rT E2f3-/-
E2f3-/-
-,
-9
p53 activity
s
9
r
--
--
:-':-
^"ONWO
M"
cdc2
' p-tubulin
3-s
4S
-
~,-"-me-
p21CIP
¢-tubulin
pa.4sagenumber
Figure 1. The effect of E2fJ3-loss on gene expression and cell cycle progression in
actively proliferating MEFs. (A) Proliferation of asynchronous wild-type [solid line]
and E2f3 knockout MEFs [dashed line] (B) 3H-thymidine incorporation of wild-type
[black bar] and E2f3 knockout MEFs [white bar] (C) FACS cell cycle analysis of wildtype [black bar] and E2f3 knockout MEFs [white bar] (D,E) Whole cell protein extracts
from wild-type and E2f3 knockout MEFs were prepared and subject to western blotting
for actin, cyclin A, p107, cdc2, p19ARF and p21CIP or electrophoretic mobility shift
assay for active p53 (F) 3T3 assay of wildtype [solid line] and E2f3 knockout MEFs
[dashed line].
entry and cell cycle progression, we examined the specific cell cycle properties of the
actively dividing cells. In pulse labeling experiments, the E2f3-'- MEFs incorporated less
3 H-thymidine
than the wildtype control, indicating that DNA replication occurred a
reduced level within the E2f3-'- population (Figure 1B). To determine whether this
represents an inability to either enter or proceed through S-phase we assessed cell cycle
distribution by FACS. Surprisingly, the E2f3 -' - MEFs displayed a similar profile to the
wild-type controls (Figure 1C). There was a subtle increase in the S-phase population,
and a corresponding decrease in the GI population, but this is not sufficient to account
for the observed reduction in 3 H-thymidine incorporation.
We therefore conclude that
E2f3-loss slows progression through multiple stages of the cell cycle. Consistent with an
overall slowing of cell cycle, E2f3-loss caused an increase in cell size that was
independent of cell cycle phase (data not shown).
The E2F family controls the expression of genes required for passage through G1,
S and G2 /M. We therefore examined the level of classic E2F-responsive genes in the
asynchronously proliferating wildtype versus E2f3-'- MEFs by western blotting (Figure
1D) and also quantitative PCR (data not shown). However, there was no detectable
difference in the expression of classic E2F target genes between wildtype and E2f3deficient MEFs. This does not rule out a role for E2f3 in the regulation of these target
genes in other settings but strongly suggests that E2f3 is not essential for the robust
expression of E2F target genes in cycling cells. We also examined the regulation of the
p19ARF-p5 3 pathway in the wildtype versus E2f3-' - MEFs.
Although E2f3-inactivation
causes a pronounced induction of pl9 ARF,p53 and p21 CIPunder conditions of mitogeninduced arrest and cell cycle re-entry (Aslanian et al., 2004), there was no detectable
114
difference in the levels of pl9ARFor the activity of p53 in cycling wildtype versus E2f3-' MEFs (Figure
E). Primary cells possess a finite replicative lifespan, ending in a
permanent arrest known as senescence, and this is determined by the levels of pl9ARF
(Kamijo et al., 1997). Comparative analysis of wildtype and E2f3 -' - MEFs in 3T3 assays
did not yield any evidence that E2f3-inactivation causes premature replicative senescence
(Figure F). Taken together, these studies suggest that E2f3-inactivation does not have a
significant impact on the expression or function of pl 9ARFin cycling cells.
To directly test whether Arf regulation makes any contribution to the
asynchronous proliferation defect of the E2f3-'- MEFs, we also compared the properties of
cycling Arf'- versus Arf'-;E2f3 -' - MEFs (Figure 2). It is well established that Arf' MEFs
undergo asynchronous proliferation at a faster rate than the wildtype controls (Kamijo et
al., 1997). Consistent with this stimulatory effect, Arf mutation also increased the
proliferation rate of the E2f3-/ - MEFs (Figure 2A). However, the difference in the
proliferation rates of Arf';E2f3 -' - versus Arf/- MEFs was identical to that observed
between E2J3/- and wildtype MEFs (labeled as AE2f3 in Figure 2A). Thus, Arfinactivation does not suppress the asynchronous proliferation defect caused by E2f3deficiency. Accordingly, Arfmutation had no detectable affect on the decreased 3Hthymidine incorporation (compare Figures B and 2B), unaltered cell cycle phasing
(compare Figures 1C and 2C), or increased cell size (data not shown) observed in E2f3/-
versus control populations. Consistent with their elevated proliferation rate, Arf-deficient
cells express increased levels of classic E2F responsive genes relative to wild-type
controls. This suggests that Arf-loss places an additional burden on E2F activation.
115
Figure 2.
A
C
B
o
.E
o
IE
i)
a)
D
DKO
. Mgll
----c
p107
cyclinA
- ""cdc2
-
-
ArP"
.E
8
-_
rs,
F
E
Arf/-
01
OC
C
e
(
11
z
DK(
actin
Figure 2. The effect of pl9Arf-deletion on E2f3 knockout phenotype in cycling MEFs.
(A) Proliferation of asynchronous pl9Arf knockout [solid line] and E2f3;pl9Arf
double knockout MEFs [dashed line] (B) 3H-thymidine incorporation of pl9Arf
knockout [black bar] and E2f3,pl9Arf
double knockout MEFs [white bar] (C) FACS
cell cycle analysis of p19Arf knockout [black bar] and E2f3;pl9Arf double knockout
MEFs [white bar] (D) Whole cell protein extracts from pl9Arf knockout and
E2f3;pl9Arf double knockout MEFs were prepared and subject to western blotting for
actin, cyclin A, p107, and cdc2 (E) 3T3 assay of pl9Arf knockout [solid line] and
E2f3;pl9Arf knockout MEFs [dashed line] (F) Low-density colony formation assay
assessing the status of E2f3 on p19Arf knockout MEFs.
Nevertheless, even in the absence of Arf, cycling E2f3 -'- MEFs did not show any
detectable defect in the expression of E2F-responsive genes as judged by either direct
examination of selected targets (Figure 2D) or micro-array analysis (our unpublished
data). Deletion of Arfis known to bypass replicative senescence (Kamijo et al., 1997).
Consistent with this notion, Arf mutation was sufficient to immortalize the E2f3 /- MEFs
(Figure 2E). However, Arf'-;E2f3 -'- MEFs proliferated at a greatly reduced rate relative to
Arf/- controls and this is difference was maintained during repeated passaging (Figure
2E). Thus, E2f3-loss does not affect the replicative lifespan of primary cells, but it results
in a stable defect in asynchronous proliferation. This proliferation defect was further
illustrated by the difference in the ability of Arf'- versus Arf'-;E2f3 -' - MEFs to form
colonies in low-density colony formation assays (Figure 2F). Taken together, these data
indicate that the asynchronous proliferation defect of the E2f3-/ - MEFs is independent of
E2f3's role in the expression of Arf
Given these observations, we conclude that E2f3 controls key cellular processes
through at least two distinct mechanisms. We have previously shown that E2f3 is
required for mitogen-induced cell cycle re-entry and genetic analysis suggests that this
reflects E2F3's role in the transcriptional repression of Arf(Aslanian
et al., 2004). The
E2f3-' - MEFs join a growing group of mutant MEFs including Bmil '- - MEFs (Jacobs et al.,
1999) whose phenotypes argue that the degree of activation of the Arf-p53 pathway is the
primary determinant of the rate of cell cycle re-entry. Importantly, Arf mutation
completely suppresses the mitogen-induced defects in both cell cycle re-entry and E2Fresponsive gene activation in the E2f3-deficient cells suggesting that both are an indirect
117
consequence of the derepression of p19Aa and activation of p53. Experiments in this
current study show that E2f3 controls the rate of asynchronous proliferation and this is
entirely Arf-independent. We anticipated that this asynchronous proliferation defect
would reflect a requirement for E2F3A in the activation of classic E2F-responsive genes.
However, cycling E2f3 -'- MEFs expressed E2F-target genes at a comparable level of
E2f3 +' + controls in either the presence or absence of Arf. Since it is difficult to observe
variations in E2F target gene expression in cycling cell populations, it is an open question
whether gene activation and/or some other function accounts for E2f3's role in
asynchronous proliferation.
E2f3 controls the rate of tumor development in an Arf-independent manner.
Cellular transformation results from the abrogation of normal checks and balances
in cell cycle regulation. We therefore wished to determine how E2f3-loss affects the
properties of transformed cells. It have previously been shown that Arf'- MEFs can be
transformed by the expression of oncogenic ras. We therefore introduced activated Hrasvl2 in the Arf;E2f3 MEFs and then examined their properties (Figure 3). First, we
cultured these cells as monolayers and compared their rate of accumulation (Figure 3A).
This analysis showed that mutation of one, then both, E2f3 alleles caused a progressively
reduction in the proliferation rate of H-rasV'2 ;Arf/-MEFs (Figure 3A). Next, we
examined the properties of the H-rasVl2;Arf- MEFs in in vitro transformation assays.
E2f3 mutation did not prevent the H-rasVl2;Arf/- MEFs from producing foci in
monolayers or clones in soft agar (Figure 3B). However, E2f3 controlled the rate of
development of these transformed foci/clones in a dose-dependent manner (Figure 3B).
118
Figure 3.
A
6)
-o
3+/-
E
3-,.s_
:
C
6)
a)
a,6
Cr
Days
B
Focus formation
WT
E2f3+/
E2f3-/-
161
14
E
E 12'
)
10
E
8
I
E6
CO
E
2'
-Li
0
WT
E2f3'/ - E2f3
Figure 3. The effect of E2f3-loss on transformed pl9Arf knockout MEFs. (A)
Proliferation of transformed E2f3,pl9Arf compound mutant MEFs [wild-type E2f3
solid line; heterozygous E2f3 dashed line; E2f3 knockout dotted line] (B) Focus
formation and soft agar growth by transformed E2f3;pl9Arf compound mutant
MEFs (C) Tumor diameter of transformed E2f3;pl9Arf compound mutant MEFs two
weeks following injection into nude mice.
To further explore this observation, we assayed these H-rasV'2 ;Arf- MEFs for
their ability to form tumors in nude mice after subcutaneous injection. Two weeks after
injection, the H-rasl
2 ;Arf
/
-
MEFs that were either E2f3 +/+, E2f3 +/' or E2f3 -' - had each
produced visible tumors (Figure 3D). However, consistent with our in vitro assays, we
observed a progressive reduction in the size of the tumor as the number of functional
E2f3 alleles was reduced from two (tumor diameter = 12.4+2.9mm) to one (tumor
diameter = 7.4+2. lmm) to none (tumor diameter = 3.5+2. mm; Figure 3D). Thus, E2f3
can affect the rate of tumor development in both an Arf-independent and cell autonomous
manner.
E2f3 acts downstream of Arf in vivo.
Taken together, our cellular studies reveal a complex interplay between E2f3 and
Arf. First, E2f3 contributes to the repression of Arf, and therefore p53, in quiescent MEFs
and this regulation is required for the appropriate kinetics of cell cycle re-entry (Aslanian
et al., 2004). In this manner, E2F3 joins a growing list of transcriptional repressors,
including Bmi1, CBX7 and Pokemon, which act as upstream regulators of Arf in MEFs
(Gil et al., 2004; Jacobs et al., 1999; Maeda et al., 2005). In the case of Bmil, the genetic
interaction with Arf has also been demonstrated in vivo; Arf-derepression accounts for the
neonatal lethality of Bmil-deficient mice that results from defects in hematopoiesis and
CNS function (Molofsky et al., 2005; Park et al., 2003). This raised the question of
whether Arf plays any role in the high incidence of embryonic/neonatal
lethality observed
in the E2f3 -/- mice. Second, our cellular studies show that E2f3 acts downstream of Arfto
120
control the proliferation rate of transformed MEFs. This raised the question of whether
E2f3 influences the development of Arf-deficient tumors in vivo.
To address these questions, we generated and analyzed E2f3;Arf compound
mutant mice. First, we intercrossed E2f3+',Arf+' - mice and determined the genotype of
the progeny at weaning (Figure 4A). As we have previously observed, the E2f3 -'- mice
were under-represented at this timepoint. Somewhat surprisingly, the mutation of the Arf
had no detectable effect on the viability of these animals. Thus, in contrast to Bmil, the
requirement for E2f3 in normal development appears to be Arf-independent.
Second, to
assess E2f3's role in tumor development, we aged a cohort of Arf'- mice that were either
wildtype, heterozygous or nullizygous for E2f3 (Figure 4B). It has previously bee
reported that Arf'- mice develop a variety of tumors, primarily lymphomas and sarcomas
(Kamijo et al., 1999; Kamijo et al., 1997). Irrespective of their E2f3 status, the mice in
our colony displayed a similar tumor spectrum (data not shown). However, E2f3-loss
impeded the development of tumors, and thereby extended lifespan, in a dose-dependent
manner (Figure 4B). These data provide definitive proof that E2f3 acts in an Arfindependent manner to control the rate of tumor developmental in vivo. Extrapolating
from our cellular studies, we conclude that this reflects E2f3's role in the controlling
asynchronous proliferation.
121
Figure 4.
A
B
WT
Arft-
A
VT
14
40
15
E2f3'-
26
61
26
E2f3-/-
04
13
04
-
1
2'
0
50
100
150
200 250
Days
300
350
400
450
500
Figure 4. In vivo interaction between E2f3 and pl9Arf. (A) Progeny of E2f3,pl9Arf
mice [wild-type
double heterozygous crosses (B) Survival of adult p9Arf-deficient
E2f3 solid black line; heterozygous E2f3 solid grey line; E2f3 knockout dashed black
line].
MATERIALS AND METHODS
Mice
E2f3;pl9Arf compound mutant animals were generated by crossing E2f3+/- and p]9Arf/- mice. Genotyping was performed as previously described (Humbert et al., 2000b;
Kamijo et al., 1997). E2f3+/-;p19Arf+/- or E2f3-/-;p19Arf-/- mice were intercrossed to
generate animals to assess viability of various genotypes and surviving animals were then
aged and monitored for tumor development.
For tumorigenicity assays, 6 to 8 week old
nude mice were subcutaneously injected with 10^6 cells near each limb and tumor
growth was monitored regularly.
Cell culture
E2f3+/-;pl9Arf-/-
mice were intercrossed and MEFs were prepared from e13.5 embryos
as previously described (Humbert et al., 2000b). For growth curves, passage 4 MEFs
were plated on 6cm dishes at 3*10A5 cells per dish. For 5 days following plating,
separate dishes were counted and cell number was recorded. For assessment of
senescence, 3T3 assays were performed as previously described (Kamijo et al., 1997).
Low-density colony formation assays were performed by seeding 10^3 cells on 10cm
dishes and staining cells after 3 weeks with Modified Wright Giemsa Stain (Sigma
WG32) to visualize colonies.
123
Retroviral infection and transformation assays
Retroviral infections were performed with pBabe-Puro and pBabe-Puro-HrasV 12 vectors
that have been previously described (Humbert et al., 2000b). Retroviral production was
performed by transfection of Phoenix cells and following infection, target cells were
selected in 2 mg/mL puromycin for 3 days. Growth assays, focus formation assays, and
soft agar assays were performed as previously described (Humbert et al., 2000b).
Western blotting and electrophoretic mobility shift assay
Passage 4 MEFs were plated on 15cm dishes at 2*10A6 cells per dish. Two days after
plating cells were harvested and total protein was isolated as previously described
(Moberg et al., 1996). Western blotting was performed with 100 tg of whole cell extract
using antibodies against actin (Santa Cruz sc-1616), cyclin A (Santa Cruz sc-596), p107
(Santa Cruz sc-318), cdc2 (Santa Cruz sc-54), p19ARF (Novus NB200-106) and p21CIP
(Santa Cruz, sc-6246). p53 gel-retardation assays were performed in the presence of
supershifting antibody using NuShift according to the manufacturer's instructions.
3H-Thymidine Incorporation
Passage 4 MEFs were plated in triplicate on 3.5cm dishes at 2* 10^5 cells per dish. One
day after plating, cells were incubated with 5 EtCi of 3H-thymidine for 1 hour. Cells were
then harvested and 3H-thymidine incorporation was measured as previously described
(Moberg et al., 1996).
124
FACS
Passage 4 MEFs were plated on 6cm dishes at 3*10^5 cells per dish. Three days after
plating (at which time cell growth is linear), cells were incubated with 10M BrdU for 1
hour. Cells were then harvested and prepared for FACS analysis.
125
References.
Aslanian, A., Iaquinta, P. J., Verona, R., and Lees, J. A. (2004). Repression of the Arf
tumor suppressor by E2F3 is required for normal cell cycle kinetics. Genes Dev 18, 14131422.
Attwooll, C., Denchi, E. L., and Helin, K. (2004). The E2F family: specific functions and
overlapping interests. Embo J 23, 4709-4716.
Blais, A., and Dynlacht, B. D. (2004). Hitting their targets: an emerging picture of E2F
and cell cycle control. Curr Opin Genet Dev 14, 527-532.
Gaubatz, S., Lindeman, G. J., Ishida, S., Jakoi, L., Nevins, J. R., Livingston, D. M., and
Rempel, R. E. (2000). E2F4 and E2F5 play an essential role in pocket protein-mediated
G1 control. Mol Cell 6, 729-735.
Gil, J., Bernard, D., Martinez, D., and Beach, D. (2004). Polycomb CBX7 has a unifying
role in cellular lifespan. Nat Cell Biol 6, 67-72.
Humbert, P. O., Rogers, C., Ganiatsas, S., Landsberg, R. L., Trimarchi, J. M., Dandapani,
S., Brugnara, C., Erdman, S., Schrenzel, M., Bronson, R. T., and Lees, J. A. (2000a).
E2F4 is essential for normal erythrocyte maturation and neonatal viability. Mol Cell 6,
281-291.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000b). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A., and van Lohuizen, M. (1999).
The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and
senescence through the ink4a locus. Nature 397, 164-168.
Kamijo, T., Bodner, S., van de Kamp, E., Randle, D. H., and Sherr, C. J. (1999). Tumor
spectrum in ARF-deficient mice. Cancer Res 59, 2217-2222.
Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing, J. R., Ashmun, R. A.,
Grosveld, G., and Sherr, C. J. (1997). Tumor suppression at the mouse INK4a locus
mediated by the alternative reading frame product p19ARF. Cell 91, 649-659.
Lowe, S. W., and Sherr, C. J. (2003). Tumor suppression by Ink4a-Arf: progress and
puzzles. Curr Opin Genet Dev 13, 77-83.
Maeda, T., Hobbs, R. M., Merghoub, T., Guernah, I., Zelent, A., Cordon-Cardo, C.,
Teruya-Feldstein, J., and Pandolfi, P. P. (2005). Role of the proto-oncogene Pokemon in
cellular transformation and ARF repression. Nature 433, 278-285.
Moberg, K., Starz, M. A., and Lees, J. A. (1996). E2F-4 switches from p130 to p107 and
pRB in response to cell cycle reentry. Mol Cell Biol 16, 1436-1449.
126
Molofsky, A. V., He, S., Bydon, M., Morrison, S. J., and Pardal, R. (2005). Bmi-1
promotes neural stem cell self-renewal and neural development but not mouse growth
and survival by repressing the pl6Ink4a and pl9Arf senescence pathways. Genes Dev 19,
1432-1437.
Muller, H., Bracken, A. P., Vernell, R., Moroni, M. C., Christians, F., Grassilli, E.,
Prosperini, E., Vigo, E., Oliner, J. D., and Helin, K. (2001). E2Fs regulate the expression
of genes involved in differentiation, development, proliferation, and apoptosis. Genes
Dev 15, 267-285.
Park, I. K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L., Morrison, S.
J., and Clarke, M. F. (2003). Bmi-1 is required for maintenance of adult self-renewing
haematopoietic stem cells. Nature 423, 302-305.
Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. (1995). Alternative reading
frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of
inducing cell cycle arrest. Cell 83, 993-1000.
Rempel, R. E., Saenz-Robles, M. T., Storms, R., Morham, S., Ishida, S., Engel, A., Jakoi,
L., Melhem, M. F., Pipas, J. M., Smith, C., and Nevins, J. R. (2000). Loss of E2F4
activity leads to abnormal development of multiple cellular lineages. Mol Cell 6, 293306.
Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A., and Dynlacht,
B. D. (2002). E2F integrates cell cycle progression with DNA repair, replication, and
G(2)/M checkpoints. Genes Dev 16, 245-256.
Storre, J., Elsasser, H. P., Fuchs, M., Ullmann, D., Livingston, D. M., and Gaubatz, S.
(2002). Homeotic transformations of the axial skeleton that accompany a targeted
deletion of E2f6. EMBO Rep 3, 695-700.
127
CHAPTER 4
Tumor development in the absence of p53
is not dependent on the regulation of proliferation by E2Jf3
Aaron Aslanian and Jacqueline A. Lees
Author's contributions: Figure 1, 2 and 3
128
ABSTRACT
The E2F transcription factor is a key regulator of cellular proliferation (Trimarchi
and Lees, 2002). E2F controls the expression of genes involved in cell cycle progression,
DNA replication and mitosis. However, E2F also has functions broader than basic cell
cycle control, such as regulation of DNA damage, apoptosis and development.
We have
previously shown that E2F3 is a critical regulator of proliferation. The loss of E2F3
results in a significant proliferation defect in mouse embryonic fibroblasts (MEFs)
(Humbert et al., 2000). E2F3 loss also impairs tumorigenesis in multiple mouse tumor
models (Ziebold et al., 2003). In this study, we examined the interaction between E2f3
and the p53 tumor suppressor protein in tumorigenesis. Unlike other mouse tumor
models, the loss of E2f3 did not impair tumor development in p53 knockout mice.
Transformed p5.3-deficient MEFs also were highly tumorigenic regardless of E2f3 status.
Nevertheless, E2f3-loss still resulted in a proliferation defect in p53-deficient MEFs.
This data indicate that E2F3 may influence tumorigenesis through mechanisms other than
cell cycle control. Additionally, E2f3 appears to function upstream (or in parallel) of p53
in tumor development while p53 functions upstream of E2f3 in cellular proliferation.
129
INTRODUCTION
Several studies have linked the deregulation of cell cycle controls to tumor
development.. Indeed, unrestrained proliferation is one of the hallmarks of cancer
(Hanahan and Weinberg, 2000). Inappropriately proliferating cells are normally
eliminated via programmed cell death, another process often targeted in cancer. The p53
tumor suppressor protein can affect both cell cycle progression and the induction of
programmed cell death through transcriptional regulation of its target genes.
Consequently, p53 is functionally inactivated in almost all tumors. Cells lacking
functional p53 are compromised in their ability to appropriately respond to various types
of cellular stress such as oncogenic challenge or DNA damage. Mouse models have
demonstrated that p53-deficiency promotes the development of a wide array of tumor
types (Attardi and Jacks, 1999).
The retinoblastoma (Rb) pathway is another critical determinant of cell cycle
progression frequently disrupted in tumors. Rb-deficiency results in inappropriate
proliferation during embryo development and tumorigenesis in adult mice (Jacks et al.,
1992; Lee et al., 1992). The retinoblastoma protein (pRb) and the related pocket proteins
p 107 and p130 control proliferation via the E2F family of transcription factors (Trimarchi
and Lees, 2002). E2F regulates the expression of target genes critical for cell cycle
progression, DNA replication and mitosis. There are currently eight genes encoding E2F
proteins, but pRb interacts specifically with E2F1-4. The generation of different Rb;E2f
compound mutant animals has demonstrated that deregulation of E2F activity represents
an important component of the inappropriate proliferation observed in development and
130
cancer (Lee et al., 2002; Tsai et al., 1998; Yamasaki et al., 1998; Ziebold et al., 2003;
Ziebold et al., 2001).
Considerable crosstalk exists between E2F and the p53 pathway. This suggests
that an important connection between regulation of cellular proliferation and p53 activity
may operate in cancer. E2F functions downstream of p53 through its most well
characterized target genes, p21CIP. Expression of p21CIP leads to the inhibition of
cyclin-cdk activity, accumulation of hypo-phosphorylated pRb, and the release of free,
active E2F. However, E2F can function upstream of p53 as well. E2F1 has been shown
to increase p53 activity through at least two mechanisms. First, E2F1 over-expression
induces expression of p19ARF, which positively regulates p53 by sequestering the
ubiquitin ligase mdm2. Second, E2F1 regulates the activity of Chk2 and ATM kinases
that are responsible for phosphorylation of p53, which also stabilizes p53 by preventing
the mdm2-p53 interaction. Several pro-apoptotic E2F target genes have also been
identified recently.
The fact that E2F functions both upstream and downstream of p53 was further
illustrated through the use of a carboxy-terminal mutant of E2F1, E2F-DB (Rowland et
al., 2002). This mutant lacks both the pocket protein binding domain and the transactivation domain of E2F1. Consequently, it cannot repress transcription through pRb
nor can it stimulate transcription.
E2F-DB retains DNA binding activity, and can
displace endogenous E2Fs from the promoters of target genes. It was shown that
expression of E2F-DB leads to activation of p53 via p19ARF. Normally, induction of
p53 activity would lead to growth arrest. However, cells expressing E2F-DB continue to
131
grow in the presence of elevated p53 activity. This is due to de-repression of E2F target
genes caused by E2F-DB.
Mouse models have been used to probe the relationship between E2F and p53 in
tumorigenesis, but have yielded conflicting results. In one study, tumor susceptibility of
p53 knockout mice was drastically reduced when both copies of E2fl were also deleted
(Wikonkal et al., 2003). A second study showed the exact opposite result, no change in
mortality between p53 knockout mice and E2fl;p53 double knockout mice (Wloga et al.,
2004). There was some alteration in tumor spectrum reported in this study. The reason
for the discrepancies between these two studies is not fully apparent. Further obscuring
the function of E2F1 in tumorigenesis is the fact that E2F1 has both oncogenic and tumor
suppressive properties (Yamasaki et al., 1996).
The specific role of other E2Fs in tumorigenesis is even less well understood.
There is also little indication how E2Fs other than E2F1 might influence p53 function.
We are particularly interested in examining the contribution of E2F3 because of its key
role in the regulation of cellular proliferation.
E2F3 DNA binding activity has been
shown increase at the G1/S transition during multiple cell cycles (Leone et al., 1998). It
specifically associates with the promoters of several E2F target genes in this phase of the
cell cycle (Giangrande et al., 2004). Mouse embryonic fibroblasts (MEFs) lacking E2f3
are severely impaired in their proliferation (Humbert et al., 2000; Wu et al., 2001).
Additionally, we have shown that E2F3 acts as an upstream regulator of the p53 pathway
via p19ARF (Aslanian et al., 2004).
We previously investigated the role of E2F3 in the tumor development of pl9Arfdeficient mice. Generalized lymphomas and soft tissue sarcomas are the predominant
132
tumors that arise in pl9Arf knockout mice. Deletion of E2f3 resulted in a dose-dependent
extension in the lifespan of pl9Arf deficient mice. This data suggested that E2F3dependent proliferation is a critical downstream component of tumor development that
occurs in the absence of p19ARF.
Because p19ARF functions upstream of p53, we reasoned that E2F3 should also
impair tumorigenesis in p53 deficient mice. However, E2f3 status had no effect on the
survival of p53 null mice. This discrepancy could not be attributed to the different types
of tumors that arise in these two mouse strains, as transformed p53 knockout MEFs also
efficiently formed tumors in nude mice regardless of E2f3 status. Since we had
previously shown that E2f3-deficiency significantly impairs cellular proliferation, we
examined the consequences of E2f3 loss in p53 knockout MEFs. Similar to our previous
observations using E2f3;pl9Arf compound mutant MEFs, proliferation of E2f3;p53
double knockout MEFs are also impaired. This indicates that the effect of E2F3 on
proliferation is not sufficient to explain its function in tumorigenesis, and E2F3 must
have other functions with the cell.
RESULTS
Generation and survival of E2f3;p53 compound mutant mice
To ascertain the interaction of E2f3 and p53 in development and tumorigenesis,
we intercrossed E2f3 and p53 mutant animals. E2f3 knockout mice are not viable in a
pure strain background, so it was necessary to use a mixture of genetic backgrounds.
Double heterozygous E2f3,p53 mice were first generated in both C57B16 and 129Sv
strain backgrounds.
These animals then were inter-bred to generate an F1 population
133
used in this study. These animals are an even mixture of two strains, which should
minimize any variation caused by genetic modifiers.
Animals were genotyped at three weeks of age to determine whether E2f3 and
p53 genetically interact to affect development. p53 status had no effect on the viability of
E2f3-deficient animals. Female specific lethality of p53 null animals has been reported
due to failure of neural tube closure. However, we did not observe any skewing of the
male to female ratio in p53 knockout mice, regardless of E2f3 status. These data suggest
that E2f3 and p53 do not genetically interact in murine development.
p53 knockout animals were aged to determine the effect of E2f3 status on tumor
development (Figure 1). p53 knockout mice with both copies of E2f3 succumbed to
tumors with a mean latency of 178 days. Animals with a single copy of E2f3 and those
with no E2f3 succumbed to tumors with a mean latency of 165 days and 189 days
respectively. Regardless of E2f3 status, p53 knockout mice develop thymic lymphomas
and soft tissue sarcomas. Thus E2f3 status has no effect on tumorigenesis in p53
knockout mice.
E2f3 loss does not impair the tumorigenicity of transformed p53-deficient MEFs
It was surprising that E2f3 status had no effect on tumorigenesis in p53 knockout
mice because the deletion of E2f3 resulted in a significant extension in the lifespan of
previously examined mouse tumor models, including pl9Arf knockout mice. To exclude
the possibility that the results obtained in this study were due to differences in the tumors
types being examined, we investigated the consequences of E2f3 loss on transformed
134
Figure 1.
1
l
~,,I~l.I1
C-~--.
I
1
III
-- E2f3 wildtype (n=23)
0.9
'_
--- E2f3 heterozygous (n=43)
La
0.8
0.7
--
E2f3 knockout (n= 13)
0.6
-
B~LS4
cU
¢
._Ct
U
0.5
0.4
1
U
0.3
U
0.2
0.1
7 i is__P
0
0
50
100
150
200
250
300
350
days
Figure 1. E2f3-loss has no effect on the survival of p53-deficient mice. p53-deficient
mice that were either E2f3 wild-type (black line with black boxes), E2f3 heterozygous
(black line with white boxes) or E2f3 knockout (grey line with grey boxes) were aged
and monitored for tumors.
p53-deficient MEFs. We have previously shown in this system that the loss of E2f3
results in a dose-dependent impairment of transformed pl9Arf-deficient MEFs.
p53 knockout and E2f3;p53 double knockout MEFs were transformed using a
constitutively active H-ras allele. The loss of E2f3 did not affect the ability of p 5 3deficient MEFs to be transformed as judged by focus formation and growth in soft agar
(Figure 2B and 2C). These cells were subcutaneously injected into nude mice, which
were then monitored for tumor formation (Figure 2D). Both genotypes rapidly gave rise
to tumors with similar growth rates. After two weeks, the mean diameter of tumors
arising from transformed p53 knockout MEFs measured 9.2 ± 2.2 mm, while the mean
diameter of tumors arising from transformed E2f3,p53 double knockout MEFs measured
7.5 + 1.6 mm in size. The loss of E2f3 results in only a modest reduction in
tumorigenicity in transformed p53-deficient MEFs.
E2f3 loss impairs the proliferation of primary and transformed p53-deficient MEFs
Previously we had demonstrated that the loss of E2f3 results in a proliferation
defect in cycling cells. This defect cannot be suppressed by deleting p]9Arf, and
consequently E2f3 loss impairs the tumorigenicity of transformed pl9Arf-deficient
MEFs. We examined the proliferation of E2f3;p53 double knockout MEFs to determine
whether the above data could be explained by the rescue of any proliferation defect in
these cells. However, E2f3;p53 double knockout MEFs clearly proliferated at a slower
rate than p53 knockout MEFs (Figure 3A). Similar results were obtained with
transformed cells (Figure 2A). These results are consistent with our observation that
136
Figure 2.
A)
6000000
B)
5000000
I)
p53 knockout rasV12
4000000
3000000
¢D
2000000
1000000
0
13
3
~~0 2
T-1I%
4
5
1%
-1
1
L ;pJ.).5 oule
knockout rasV 12
days
- -- p53 knockout rasV12
-- -- E2f3;p53 double knockout rasV12
C)
p53 knockout
D)
A
IL-.J -
rasV 12
12.0 a)
10.0 -
T
8.0 c'
E
E2f3,p53 double
knockout rasV 12
T
6.0 4.0
0
2.0
0.0
.
p53 knockout
rasV12
E2f3;p53
double
knockout
rasV 12
Figure 2. The effect of E2J3-loss on transformed p53 knockout MEFs.
(A)
Proliferation of transformed E2f3;p53 compound mutant MEFs [wild-type
E2f3 solid
line; E2f3 knockout dashed line] (B) Growth in soft agar by transformed
E2f3;p53
compound mutant MEFs (C) Focus formation by transformed E2f3;p53
compound
mutant MEFs (D) Tumor diameter of transformed E2f3;p53 compound mutant
MEFs
two weeks following injection into nude mice.
,
Figure 3.
6000000
A)
B)
5000000
oC.
J
S ,4000000
Q,*
3000000
10
E
r.,
V -
2000000
I
C.
I
J
jrfl
--
1000000
0
0
2
1
4
3
A
days
=
Moo-n
-mIuII-
E2f3;p53 double knockout
- -I12
C)
10
cu
8
6
r=.
c
-
4
2
0
4
5
6
7
8
9
10
11
passage number
-
dRWp107
-
p53 knockout
p53 knockout - -- -E2f3;p53 double knockout
actin
cyclin A
cdc2
Figure 3. continued
D)
11. )
.)
.
T
1
..
© o 0.8 4.a r.-
T
0.6 -
)Co 0.4 Ct
0.2 -
C)
;-.4~-
0-
I
p5 3
knockout
E2f3 ;p53
double
F)
p53 knockout
knockout
E)
Qn
c)
.o
ct
45~
PU
70 60 50 -
E2f3;p53
double knockout
40 30 20 10 -
0-
G1
*p53 knockout
S
G2/M
oE2f3;p53 double knockout
Figure 3. The effect of p53-deletion on E2f3 knockout phenotype in cycling MEFs. (A)
Proliferation of asynchronous p53 knockout [solid line] and E2f3;p53 double knockout
MEFs [dashed line] (B) Whole cell protein extracts from p53 knockout and E2f3;p53
double knockout MEFs were prepared and subject to western blotting for actin, cyclin A
(sc-596), p107 (sc-318), and cdc2 (sc-54) (C) 3T3 assay of p53 knockout [solid line] and
E2f3;p53 knockout MEFs [dashed line] (D) 3H-thymidine incorporation of p53 knockout
and E2f3;p53 double knockout MEFs (E) FACS cell cycle analysis of p53 knockout
[black bar] and E2f3;p53 double knockout MEFs [white bar] (F) Low-density colony
formation assay assessing the status of E2f3 on p53 knockout MEFs.
there is no significant change in p53 activity in E2f3-deficient MEFs. The impaired
proliferation of E2f3;p53 double knockout MEFs is stably maintained over multiple
population doublings as judged by 3T3 assay (Figure 3C).
Further characterization of E2f3;p53 double knockout MEFs showed comparable
E2F target gene expression relative to p53 knockout MEFs (Figure 3B). The relative
DNA replication rates was assessed by measuring 3H-thymidine incorporation. E2f3,p53
double knockout MEFs showed only 59 percent the level of 3H-thymidine incorporation
as p53 knockout MEFs, consistent with their slower proliferation rate (Figure 3D). Cell
cycle FACS analysis of p53 knockout and E2f3;p53 double knockout MEFs yielded
similar cell cycle distribution profiles (Figure 3E). This is consistent with our previous
observations that E2f3-loss results in a slowing of the entire cell cycle.
p53-loss rescues low density growth of E2J3-deficient cells
To elucidate the function of E2F3 relevant for modulation of tumorigenicity, we
next examined the affect of E2f3-loss on low-density colony formation. p53-deficient
cells are capable of growing at low density, and when seeded at the level of individual
cells will give rise to colonies. We had previously observed that the proliferation defect
caused by the loss of E2f3 increased in severity at lower cell densities. Therefore, we
examined the role of E2f3 status on the low-density colony formation ability of p 5 3 deficient MEFs. p53-deficient MEFs efficiently formed colonies at low density,
regardless of E2f3 status (Figure 3F). This is in contrast to pl9Arf-deficient
which also form colonies, but lose this ability in absence of E2f3.
140
MEFs,
DISCUSSION
In this study, we demonstrate that the loss of E2f3 does not adversely affect
tumorigenesis that occurs in a p53-deficient setting. We first showed this result using
p53 knockout mice, which spontaneous develop thymic lymphomas and soft tissue
sarcomas. We also demonstrated that p53 knockout MEFs transformed with a
constitutively active allele of H-ras also form tumors regardless of E2f3 status. Despite
the fact that E2J3 status had no effect on tumorigenesis arising in p53-deficient cells,
E2f3-loss was still capable of impairing proliferation of both primary and transformed
p53-deficient cells. This indicates that E2F3 affects tumorigenesis through additional
mechanisms beyond the regulation of proliferation. It also suggests that E2F3 may affect
tumorigenesis through a mechanism that is p53-dependent, although downstream of p53
itself.
The fact that E2f3-loss can impair the proliferation of p53-deficient cells without
significantly impacting their ability to form tumors provides some clue as to the
mechanism through which the loss of p53 contributes to tumorigenesis. The loss of p53
relieves the inhibition of cell cycle progression and abrogates the induction of apoptosis.
Both cellular proliferation pathways and apoptotic pathways are targeted by cancer.
These experiments suggest that the loss of p53 has a more significant contribution to
apoptotic pathways than proliferative pathways. However, an alternative explanation is
that proliferative signals in the tumor are able to overcome the loss of E2f3 in ways that
do not occur in cell culture.
The suggestion that E2f3-loss does not affect tumorigenesis contradicts earlier
work, indicating that the reason for this difference must either be genetic or cell type
141
specific. One explanation would be that the cells giving rise to tumors in p53 knockout
mice do not express E2F3. However, this is unlikely since E2F3 mRNA and protein are
present in thymic lymphomas (data not shown) and soft tissue sarcomas are observed in
pl9Arf knockout mice, where the loss of E2f3 does have an impact. The experiments
with transformed MEFs also suggest this explanation is not likely to be correct. E2F3
may be having cell non-autonomous affects on tumorigenesis, but the transformed MEF
experiments make this unlikely as well. A more plausible explanation is that the loss of
E2f3 intersects with a pathway downstream of p53. Thus, the loss of p53 would prevent
E2f3 from affecting tumorigenesis.
One mechanism by which E2F3 could impact tumorigenesis is through the
regulation of genomic stability. Previous studies suggest that the loss of E2f3 leads to
errors in centrosome duplication. Proper centrosome regulation is important to ensure the
proficient segregation of DNA during mitosis. Centrosomal abnormalities are frequency
associated with cancer, and represent a mechanism by which additional mutation can
occur in cancer. Normally, errors in centrosome duplication would lead to DNA damage
and signal to p53. These cells would likely be eliminated. However, this would not be
expected to occur in p53 deficient cells, and in fact, p53-deficiency
has been reported to
promote centrosome abnormalities.
The experiments in this study also show that p53 loss permits E2f3-deficient cells
to form colonies when plated at low density. This is in contrast to p]9Arf deficient cells,
which require E2f3 to form colonies at low density. This phenotype correlates with the
effect of E2f3 on tumorigenesis and may possibly be related. Unfortunately, the
142
mechanistic basis for low-density growth is not known. It is likely that this involves
some kind of cell-signaling events and this requires further investigation.
Previous mouse tumor studies using Rb heterozygous mice did not find a
connection between E2F3 and p53. In these mice, the loss of E2f3 suppresses pituitary
tumors, but enhances the development of thyroid tumors. Because p53 loss is a common
contributing factor to the development of thyroid tumors, p53 activity was examined.
This study found no consistent difference in p53 activity due to the status of E2f3. This
would argue that E2F3 does not directly target p53 and is in fact consistent with the
observation that p53 activity is not altered in E2f3-deficient MEFs. If E2f3 loss affects
the p53 pathway downstream of p53 itself, this would explain the observation that p53
activity was unaffected in the tumors of these mice.
Inactivation of p53 occurs through a variety of mechanisms. Although initial
studies regarding p53 tumor suppressor function in mouse models have utilized germline
deletions, it is true that in human cancers, point mutations represent a frequent
mechanism of p53 inactivation.
To this end, point mutant p53 mice have been developed
and show some different properties compared to germline deletion in their cellular stress
response and tumor suppressive properties. However, experiments with E2F3 indicate
that there is a significant difference between no p53 activity and low p53 activity as
indicated by this study and similar work with p19ARF. Expression of a dominant
negative p53 allele in the background of wild-type p53 appears to also be insufficient to
provide the same response as a complete p53 null.
The fact that E2f3-loss has a different effect in the background of pl9Arfdeficiency and p53-deficiency
is not entirely surprising. There is mounting evidence to
143
suggest that p19ARF and p53 have many independent functions.
It is likely that the loss
of E2f3 impacts one of these functions during tumorigenesis, rather than a common
pathway. Additionally, direct comparison of pl9ARF and p53 in different assays suggest
that the loss of p53 produces a stronger phenotype than the loss of pl9Arf.
because some p53 activity remains in pl9Arf-deficient
pl9ARF-independent
This may be
cells. Or it may be because
pathways can activate p53.
Differences between p19ARF and p53 have also been uncovered in several tumor
models. Mice lacking either pl9Arf or p53 develop a slightly different spectrum of
tumors. Additionally, p19Arf;Mdm2;p53 triple knockout mice develop different tumors
than Mdm2;p53 double knockout mice, demonstrating that the loss of these proteins are
not entirely redundant. Crosses with other mouse models have also yielded differing
results.
Because E2F activity is commonly deregulated in most cancers, understanding
how specific E2Fs modulate tumor phenotypes will represent an important future goal.
This study uncovers two important observations regarding the role of E2F3 in
tumorigenesis.
First, although E2F3 is a critical regulator of cellular proliferation, it has
other functions that may be more relevant in the context of cancer. Second, the role
played by E2F3 in tumors is highly p53-dependent. This suggests that therapeutic
inactivation of E2f3 in tumors will only be beneficial in those that retain p53 activity.
144
MATERIALS AND METHODS
Mice
E2f3;p53 compound mutant animals were generated by crossing E2f3+/- and p53-/- mice
in either a pure C57B16 strain background or a pure 129Sv strain background.
Genotyping was performed as previously described. Pure C57B16 and pure 129Sv
E2f3+/-;p53+/- mice were intercrossed to generate F1 animals that were a 50:50 mix of
these two strain backgrounds. The viability of various genotypes was assessed and
surviving animals were then aged and monitored for tumor development. For
tumorigenicity assays, 6 to 8 week old nude mice were subcutaneously injected with
10^6 cells near each limb and tumor growth was monitored regularly.
Cell culture
E2f3+/-,p53-/- mice were intercrossed and MEFs were prepared from e13.5 embryos as
previously described. For growth curves, passage 4 MEFs were plated on 6cm dishes at
3* 10^5 cells per dish. For 5 days following plating, separate dishes were counted and
cell number was recorded. For assessment of senescence, 3T3 assays were performed as
previously described. Low-density colony formation assays were performed by seeding
10^3 cells on 10cm dishes and staining cells after 2 weeks to visualize colonies.
Retroviral infection and transformation assays
Retroviral infections were performed with pBabe-Puro and pBabe-Puro-HrasV 12 vectors
that have been previously described. Retroviral production was performed by
transfection of Phoenix cells as previously described and following infection, target cells
145
were selected in 2 mg/mL puromycin for 3 days. Growth assays, focus formation assays,
and soft agar assays were performed as previously described.
Western blotting and electrophoretic mobility shift assay
Passage 4 MEFs were plated on 15cm dishes at 2* 10^6 cells per dish. Two days after
plating cells were harvested and total protein was isolated as previously described.
Western blotting was performed with 100 tg of whole cell extract using antibodies
against actin, cyclin A, p107, cdc2, p19ARF, and p21CIP.
3H-Thymidine Incorporation
Passage 4 MEFs were plated in triplicate on 3.5cm dishes at 2*10A5 cells per dish. One
day after plating, cells were incubated with 5 tCi of 3H-thymidine for 1 hour. Cells were
then harvested and 3H-thymidine incorporation was measured as previously described
(Moberg et al., 1996).
FACS
Passage 4 MEFs were plated on 6cm dishes at 3*10A5 cells per dish. Three days after
plating (at which time cell growth is linear), cells were incubated with 10tM BrdU for 1
hour. Cells were then harvested and prepared for FACS analysis as previously described.
146
References.
Aslanian, A., laquinta, P. J., Verona, R., and Lees, J. A. (2004). Repression of the Arf
tumor suppressor by E2F3 is required for normal cell cycle kinetics. Genes Dev 18, 14131422.
Attardi, L. D., and Jacks, T. (1999). The role of p53 in tumour suppression: lessons from
mouse models. Cell Mol Life Sci 55, 48-63.
Giangrande, P. H., Zhu, W., Rempel, R. E., Laakso, N., and Nevins, J. R. (2004).
Combinatorial gene control involving E2F and E Box family members. Embo J 23, 13361347.
Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, M. A., and Weinberg, R.
A. (1992). Effects of an Rb mutation in the mouse. Nature 359, 295-300.
Lee, E. Y., Cam, H., Ziebold, U., Rayman, J. B., Lees, J. A., and Dynlacht, B. D. (2002).
E2F4 loss suppresses tumorigenesis in Rb mutant mice. Cancer Cell 2, 463-472.
Lee, E. Y., Chang, C. Y., Hu, N., Wang, Y. C., Lai, C. C., Herrup, K., Lee, W. H., and
Bradley, A. (1992). Mice deficient for Rb are nonviable and show defects in neurogenesis
and haematopoiesis. Nature 359, 288-294.
Leone, G., DeGregori, J., Yan, Z., Jakoi, L., Ishida, S., Williams, R. S., and Nevins, J. R.
(1998). E2F3 activity is regulated during the cell cycle and is required for the induction
of S phase. Genes Dev 12, 2120-2130.
Moberg, K., Starz, M. A., and Lees, J. A. (1996). E2F-4 switches from p130 to p107 and
pRB in response to cell cycle reentry. Mol Cell Biol 16, 1436-1449.
Rowland, B. D., Denissov, S. G., Douma, S., Stunnenberg, H. G., Bernards, R., and
Peeper, D. S. (2002). E2F transcriptional repressor complexes are critical downstream
targets of p19(ARF)/p53-induced proliferative arrest. Cancer Cell 2, 55-65.
Trimarchi, J. M., and Lees, J. A. (2002). Sibling rivalry in the E2F family. Nat Rev Mol
Cell Biol 3, 11-20.
Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L., and Jacks, T. (1998).
Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends
survival of Rb-deficient mouse embryos. Mol Cell 2, 293-304.
147
Wikonkal, N. M., Remenyik, E., Knezevic, D., Zhang, W., Liu, M., Zhao, H., Berton, T.
R., Johnson, D. G., and Brash, D. E. (2003). Inactivating E2f reverts apoptosis
resistance and cancer sensitivity in Trp53-deficient mice. Nat Cell Biol 5, 655-660.
Wloga, E. H., Criniti, V., Yamasaki, L., and Bronson, R. T. (2004). Lymphomagenesis
and female-specific lethality in p53-deficient mice occur independently of E2fl. Nat Cell
Biol 6, 565-567; author reply 567-568.
Wu, L., Timmers, C., Maiti, B., Saavedra, H. I., Sang, L., Chong, G. T., Nuckolls, F.,
Giangrande, P., Wright, F. A., Field, S. J., et al. (2001). The E2F1-3 transcription factors
are essential for cellular proliferation. Nature 414, 457-462.
Yamasaki, L., Bronson, R., Williams, B. O., Dyson, N. J., Harlow, E., and Jacks, T.
(1998). Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rbl(+/-)mice.
Nat Genet 18, 360-364.
Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996).
Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85, 537-548.
Ziebold, U., Lee, E. Y., Bronson, R. T., and Lees, J. A. (2003). E2F3 loss has opposing
effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but
metastasis of medullary thyroid carcinomas. Mol Cell Biol 23, 6542-6552.
Ziebold, U., Reza, T., Caron, A., and Lees, J. A. (2001). E2F3 contributes both to the
inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev
15, 386-391.
148
CHAPTER
5
DISCUSSION
149
The Rb pathway and the p53 pathway are often disrupted in tumors. Both provide
critical regulation of S-phase entry and progression. The Rb pathway regulates cellular
proliferation in response to growth factor signaling whereas the p53 pathway is
specifically activated in response to various forms of cellular stress. Despite being
considered as separate pathways, there is considerable crosstalk between components of
these two pathways.
My studies have focused on the genetic interactions between one member of the
E2F family of transcription factors, E2F3, and p19ARF/p53. I have explored these
interactions in multiple settings and my studies have shown that their relationship is
highly context dependent. In Chapter 2, I show that E2F3 is unique in its ability to bind
to the pl9Arf promoter in normal cells. In E2f3 knockout MEFs, pl9ARF expression is
de-repressed during cell cycle re-entry leading to activation of p53. Analysis of
E2f3,;p9Arf as well as E2f3;p53 compound mutant MEFs demonstrate that p19ARF/p53
significantly contribute to the cell cycle re-entry defect. In Chapter 3, I examined the
contribution of p19ARF to the proliferation defect in cycling E2f3 knockout MEFs. In
Chapter 4, I examined the contribution the effect of E2f3 loss on p53 null transformed
cell and tumors. The data in Chapter 3 and 4 clearly establish that E2F3 regulates
proliferation through multiple mechanisms. In cycling cells, E2F3 regulates proliferation
through a pl9ARF/p53-independent mechanism. Transformation and tumor studies
demonstrate that the effect of E2f3-loss is dependent on the underlying genetic alterations
in the cells.
These studies stress the fact that E2F3 regulates cellular proliferation through
multiple mechanisms. In the following discussion, I will begin by giving an overview of
150
other recent studies that have sought to determine the importance of cell cycle regulators
during cell cycle re-entry and in asynchronous proliferation.
Next, I will discuss the
mechanisms by which E2F3 controls cellular proliferation, including regulation of
p19ARF and classic E2F target genes. Finally, I will discuss the potential roles for
E2F3A and E2F3B in these processes.
S-PHASE: CELL CYCLE RE-ENTRY VERSUS THE PROLIFERATION OF
CYCLING CELLS
My experiments join a growing list of studies characterizing cells deficient for
key cell cycle regulators. These studies have been critical in testing our assumptions
about the role of these proteins and the essential nature of their functions.
Recent studies
using MEFs deficient for different cyclins and cyclin dependent kinases reinforce my
conclusion that there are key differences between cell cycle re-entry and proliferation in
cycling cells. As explained below, the function of these cell cycle regulators appears to
be most critical during cell cycle re-entry.
D-type cyclins were believed to be critical for growth factor stimulated
proliferation.
However, MEFs lacking all three D-type cyclins proliferate at only a
slightly lower rate compared to wild-type MEFs and express normal levels of other cell
cycle regulators (Kozar et al., 2004). Overall phosphorylation levels of pRb and the other
pocket proteins were reduced, although two sites presumed to be D-type cyclin specific
were capable of being phosphorylated. Quiescent D-type cyclin triple mutant MEFs also
re-enter the cell cycle normally, although they display a slightly higher serum
requirement. Cdk2 kinase activity was normal as well. Previous studies had suggested
151
that D-type cyclins were necessary to sequester members of the CIP/KIP family away
from cdk2. Similar experiments utilizing MEFs deficient for cdk4 and cdk6
demonstrated that these MEFs are able to proliferate and re-enter the cell cycle from
quiescence (Malumbres et al., 2004). It is likely that because of the critical nature of
these proteins, there is selective pressure to balance their loss with compensatory
mechanisms t:o restore cell cycle progression to a viable output. Perhaps future
experiments using conditional deletion of these cell cycle regulators will uncover a more
profound phenotype.
E-type cyclins were also believed to perform several important functions required
for cell cycle progression. Surprisingly, MEFs deficient for both E-type cyclins
proliferate only slightly slower than wild-type MEFs (Geng et al., 2003; Parisi et al.,
2003). Phosphorylation of pRb appears unaffected by deletion of the E-type cyclins,
although individual sites were not examined. Expression of other components of the cell
cycle machinery was also unchanged. No gross centrosomal abnormalities were
observed either. There was a much more profound affect on the ability of E-type cyclin
deficient MEFs to re-enter the cell cycle from quiescence. Phosphorylation of the
retinoblastoma protein appears to be compensated for by A-type cyclins. Expression of
several E2F targets also appears normal. However, members of the Pre-RC such as
Mcm2, Orc2, and Cdc6 are not properly loaded in E-type cyclin deficient MEFs. These
proteins are displaced from chromatin in quiescent cells, but not in actively cycling cells,
which may explain the difference in phenotypes that was observed. Cdk2-deficient
MEFs display similar phenotypes although they showed only a mild cell cycle re-entry
phenotype (Berthet et al., 2003; Ortega et al., 2003).
152
These studies using MEFs deficient for E-type cyclins or cdk2 are consistent with
my observations showing that the regulation of E2F target gene expression (and p19ARF)
by E2F3 is most critical during cell cycle re-entry. The defect in cell cycle re-entry due
to E2f3-loss is characterized by both impairment of E2F target gene expression and
deregulation of p19ARF and p53. On the other hand, distinct mechanisms appear to be
more critical for the regulation of cellular proliferation in cycling cells. The loss of E2f3
causes a proliferation defect in cycling cells without an observable effect on classic E2F
target gene expression or p19ARF/p53. Additionally, the loss of pl9Arf orp53 fails to
suppress this defect. Together, these studies demonstrate that E2F3 regulates
proliferation through multiple mechanisms. The following sections discuss how the
regulation of p19ARF and classic E2F target genes impact cellular proliferation.
E2F3 REGULATION OF THE p53 PATHWAY THROUGH pl9Arf AND ITS
ROLE IN PROLIFERATION
Previous studies have identified a role for E2F in pl9Arf regulation. Initial
evidence for this came from experiments showing that E2F1 over-expression was capable
of inducing ARF expression. The Arfpromoter contains multiple E2F consensus binding
sites and is E2F responsive (Bates et al., 1998). However, these studies did not explain
how endogenous expression of p19ARF was normally regulated and whether E2F also
played a role in this process. A more recent study suggested that p19ARF expression
might be regulated by E2F mediated repression, but again did not address which
endogenous E2Fs might function in this regard (Rowland et al., 2002). The fact that
pl9ARF does not behave as a classic E2F target gene suggests that a novel mode of
regulation might be involved in its regulation.
153
We demonstrate that E2F3 is the sole E2F family member that normally regulates
endogenous p19ARF expression in primary cells. In the absence of E2f3, p19ARF
expression is de-repressed in quiescent cells as well as during cell cycle re-entry.
Expression of p19ARF in cycling cells is unaffected by the loss of E2f3, suggesting that
p19ARF may be growth regulated even if it is not cell cycle regulated. The ability of
E2F3 to regulate pl9Arf expression, and therefore p53 activity, may represent a major
arm of E2F cell cycle control. This data also raises additional questions about the
mechanism through which E2F3 regulates pl9Arf and how the function of E2F3 relates
to other known regulators of pl9Arf.
Regulation of pl9Arf by E2F
Repression of pl9Arf by E2F3 appears to be necessary to decrease levels of
p19ARF when cells are deprived of serum and enter quiescence. This raises the question
of why repression of p19ARF is necessary in quiescent cells and why it should require
regulation by E2F. One explanation is that repression of p19ARF during quiescence is
necessary to prevent the onset of cellular senescence. Induction of pl9Arf is observed as
cells undergo senescence. This permanent form of growth arrest bears some similarities
to quiescence but also some important differences. One critical distinction is that cells
can come out of quiescence to resume proliferation while senescence is an end state of
growth arrest. Expression of pl9Arf may serve as one mechanism through which cells
distinguish these cell cycle states. As E2F activity is altered in response to both
quiescence and senescence, it a good candidate for implementing these different
transcriptional programs.
154
Another explanation for the repression of pl9Arf during quiescence is that it is a
result of pl9Arf regulation by growth signals. As a downstream component of the Rb
pathway, E2F activity is certainly regulated by growth factor signaling. Hyper-
proliferative signals are known to induce expression of p9Arf.
Perhaps normal
proliferative signals are responsible for the basal level of p19ARF expression found in
normally cycling cells. Quiescent cells, deficient for mitogenic signals, would not
display any p9,Arf expression. This would imply that the loss of E2f3 does not results in
de-repression of p19ARF, but rather results in incomplete cell cycle arrest, and the
residual proliferative signaling is sufficient to maintain basal p19ARF expression.
Consistent with this hypothesis, I have shown that E2f3-deficient MEFs possess a cell
cycle exit defect.
A second question raised by these observations is how the cell distinguishes
oncogenic activation responsible for induction of pl9ARF from E2F activation required
for cell cycle progression, especially during cell cycle re-entry. One possibility is that
oncogenic activation leads to post-translational modification of E2F. This could
represent a modification of E2F3B that would render it functionally inert. Or, more
likely, this would be a modification of the activating E2Fs, allowing them to bind to the
p19ARF promoter. Post-translational modification of transcription factors has been
shown to alter their activity and in some cases even provide functional specificity. The
identification of post-translational modifications on the E2Fs, and how they change in
different settings, will be instrumental in testing the hypothesis.
A final question raised by these studies is the issue of promoter occupancy with
regard to E2F regulation of pl9ARF during normal and oncogenic conditions. E2F3 is
155
observed to be present at the pl9Arf promoter throughout the cell cycle as well as during
oncogenic challenge. This would indicate that activation of pl9Arf occurs even in the
presence of E2F3. There are multiple E2F sites in the pl9Arf promoter, which offers one
explanation for this observation. If activation and repression of pl9Arf utilize different
E2F binding sites, activation of pl9Arf could occur through the second site presuming
that it is dominant over repression.
However, an equally likely explanation is that within
the population, some cells are repressing p]9Arf while others have activated pl9Arf
expression. Another possibility is that E2F3, although present at the promoter, is not
functional due to a post-translational modification or a missing cofactor. Mutational
analysis of these E2F binding sites have not proven informative in reporter gene assays,
meaning that knock-in of mutant E2F consensus binding sites into the p19Arf promoter
may be the only way to directly address this question.
Other regulators of pl9Arf
Thus far, the mechanism of E2F3 regulation of p]9Arf remains obscure. No
pocket proteins have been found at the p19Arf promoter, but it has been difficult to place
pRb at the promoter of many of its putative target genes. Several other genes have been
identified as regulators of p19Arf, including AP-1, TBX2, TBX3, Pokemon, CBX7, and
Bmil (Ameyar-Zazoua et al., 2005; Brummelkamp et al., 2002; de Stanchina et al., 1998;
Dimri et al., 2000; Jacobs et al., 2000; Jacobs et al., 1999; Lingbeek et al., 2002;
Molofsky et al., 2005; Palmero et al., 1998; Park et al., 2003; Zindy et al., 1998).
However, these studies have done little to clarify the relationship of these different
proteins or the biological significance of their regulation of pl9Arf.
156
The existence of multiple regulators of p19Arf is useful in explaining the complex
regulation of this gene. Clearly, there are different requirements for pl9Arf regulation in
cycling cells as opposed to during cell cycle re-entry. The p53 pathway is known to be
important for establishing quiescence, but the role of p19ARF has not been examined
(Itahana et al., 2002). E2F3 is essential for pl9Arf repression in quiescent MEFs, but not
in cycling MEFs. One explanation for this distinction may involve E2F3 cofactors
required for repression of pl9Arf. If these proteins are only expressed in quiescent cells,
or only interact with E2F3 at this time, this could explain the observations made in E2f3-
deficient MEFs. One prediction from this hypothesis would be that cells lacking these
cofactors would display similar deregulation of pl9Arf during cell cycle re-entry, but
normal expression in cycling cells.
Another explanation for the complex regulation of pl9Arf requires that a second,
E2F3-independent complex bind the p19Arf promoter in cycling cells. E2F3 might also
have a role in the regulation of pl9Arf in cycling cells, but repression of pl9Arf
expression by E2F3 in this setting would be redundant. This model would predict that
only the inactivation of both complexes would result in deregulation of pl9Arf in this
setting. Additionally, this second complex would have to be inactive in quiescent cells,
so that E2F3 alone would be responsible for repression of pl9Arf in this context.
Studies of the other p19Arf regulators support the model that they function
independently of E2F3. MEFs deficient for Bmi-1, junD, or Pokemon display
deregulation of p19ARF and/or p53 cycling cells (Jacobs et al., 1999; Maeda et al., 2005;
Weitzman et al., 2000). Deletion of pl9Arfor p53 suppresses the proliferation defect.
These regulators, unlike E2F3, are clearly required for proper regulation of the p53
157
pathway in cycling cells. MEFs deficient for Bmi-I, junD, or Pokemon have not been
examined for a cell cycle re-entry phenotype.
Atm-deficient MEFs display many phenotypes in common with E2f3-deficient
MEFs including a cell cycle re-entry defect, proliferation impairment, and increased cell
size (Kamijo et al., 1999; Xu et al., 1998). Expression of pl9ARF is also elevated,
although this was observed in cycling cells. Deletion of either p]9Arf, p53, or p21Cip
has been shown to suppress the cell cycle re-entry defect caused by Atm-loss.
Asynchronous proliferation and cell size are also improved, but not fully rescued.
Interestingly, p21CIP protein but not mRNA is elevated in Atm-deficient cells, suggesting
that the stability of this protein is altered in these cells.
Additional experiments will be necessary to place the function of E2F3 along with
these other pl9Arfregulators.
Some of these other regulators may directly control pl9Arf
expression at the promoter, and may also interact with E2F3. This could be tested
biochemically by identifying protein-protein interactions, or genetically through
functional epistasis.
Contribution of p21CIP to p19ARF/p53 deregulation and its role in proliferation
Although p19ARF and p53 possess multiple independent functions, that fact that
the loss of either is sufficient to alleviate the cell cycle re-entry defect in E2J3-deficient
MEFs suggests that p19ARF and p53 are functioning in a common pathway to regulate
cell cycle re-entry. The most well-characterized mechanism of cell cycle regulation by
the p53 pathway is through one of its transcriptional targets, the cyclin dependent kinase
inhibitor p21CIP (Bunz et al., 1998; el-Deiry et al., 1993; Niculescu et al., 1998). MEFs
lacking p21Cip show augmented proliferation similar to cells lacking either pl9Arf or
158
p53 (Brugarolas et al., 1995; Deng et al., 1995). To ascertain whether p21CIP regulation
is the mechanism responsible for this phenotype, compound mutant E2f3;p21Cip MEFs
could be generated, and quiescent double knockout MEFs could be tested for their ability
to re-enter the cell cycle. The levels of p21CIP itself are deregulated in E2f3-deficient
cells during cell cycle re-entry, which further supports the likelihood that p21CIP is
involved.
Regulation of p21CIP is complex and involves both transcriptional and posttranscriptional mechanisms (Sherr and Roberts, 1999). These studies suggest that
additional mechanisms besides p53 activation contribute to the up-regulation of p21CIP.
E2f3,;p9Arf double knockout MEFs still show a subtle increase in p21CIP protein levels
despite the fact that p53 activity is restored to normal. Additionally, p21CIP protein
levels are slightly elevated in cycling E2f3-deficient MEFs, despite normal p19ARF
expression and normal p53 activity. There are many possible mechanisms that could
account for this alteration in p21CIP expression. The stability of p21CIP protein is
regulated via the ubiquitin-mediated proteasome degradation pathway in a cell cycledependent manner (Bornstein et al., 2003). Additionally, E2F has been suggested to
transcriptionally regulate p21CIP expression (Hiyama et al., 1998; Radhakrishnan et al.,
2004).
To better understand the role played by p21CIP, changes in its mRNA and protein
should be more closely examined. Examination of p21CIP mRNA in E2f3;pl9Arf
double knockout MEFs will reveal whether the residual change in p21CIP levels is
transcriptional. Similar comparisons can be made between cycling wild-type and E2f3
knockout MEFs. If transcription of p21CIP is specifically deregulated, this may indicate
159
a direct role for E2F3. Expression of p21CIP may be coordinately regulated by E2F and
p53, which can be examined by ChIP. Alternatively, if p21CIP transcript levels are
normal, this would indicate a post-transcriptional mechanism of p21CIP regulation by
E2F3. It is likely that multiple mechanisms influence p21CIP levels in E2J3-deficient
MEFs.
Although p21CIP is the most extensively examined p53 target gene involved in
mediating cell cycle arrest, there are multiple p53 target genes that have been shown to be
important for p53 to inhibit cell cycle progression (Iliakis et al., 2003; Taylor and Stark,
2001). If evidence of transcriptional regulation is found in E2f3 knockout MEFs, other
p53 target genes should be monitored to determine whether they are inappropriately
elevated as well., Apoptotic targets of p53 such as Bax and Perp should also be
monitored to see whether E2F3 affects the expression of p53 target genes involved in
apoptosis (Fridman and Lowe, 2003). As yet, there is no evidence for any significant
change, positive or negative, in the apoptotic response of E2f3-deficient cells. However,
this may merely represent that the appropriate conditions have not been examined. For
example, E2f3-deficient cells may be more sensitive to specific types of cellular stress.
E2F3 REGULATION OF E2F TARGET GENES AND THEIR ROLE IN
PROLIFERATION
The importance of E2F in cell cycle progression is highlighted by early studies
with adenovirus, in which quiescent cells are stimulated to enter S-phase via deregulation
of E2F activity. The identification of classic E2F target genes fit well with this function,
as many of them play key roles in promoting cell cycle progression and DNA replication.
160
However, several years later we are still trying to understand the different mechanisms
through which E2F can regulate proliferation and what roles are played by specific E2Fs.
Several studies have specifically indicated that E2F3 plays a critical role in the
regulation of these genes. E2F3 can be found at the promoter of many classic E2F target
genes by ChIP (Giangrande et al., 2004). The fact that deregulation of classic target gene
expression was observed in E2f3 knockout MEFs during cell cycle re-entry also
confirmed the idea that E2F3 controls proliferation through its regulation of classic E2F
target genes (Humbert et al., 2000). However, the regulation of pl9ARF/p53 by E2F3
requires the role of E2F3 in the direct regulation of classic E2F target gene expression to
be re-examined.
E2F3 control of proliferation through classic E2F target gene regulation
Clearly, the expression of classic E2F target genes, many of which are critical for
cell cycle progression and DNA progression, is essential for S-phase entry. However, it
is no longer clear whether E2F3 is essential to accomplish the appropriate expression of
E2F target genes in either cycling cells or during cell cycle re-entry. In cycling E2f3deficent MEFs, classic E2F target genes are normally expressed. During cell cycle re-
entry, the observed impairment in the induction of classic E2F target gene expression
could be solely attributed to deregulation of p19ARF/p53 because the loss of either
suppresses the cell cycle re-entry defect and rescues classic E2F target gene expression.
To address these issues, p19ARF/p53 could be knocked down to wild-type levels
(but not completely eliminated) in E2f3-deficient cells using shRNA vectors. Cell cycle
re-entry kinetics can be monitored in these cells, as well as the induction of E2F target
genes. There are many possible outcomes from this experiment. If the cell cycle re-entry
161
defect were corrected, this would indicate that the p53 pathway is the primary mechanism
through which E2F3 regulates cell cycle re-entry. It may also be possible to test this in
either E2f3a or E2f3b specific knockout MEFs, if the regulation of the p53 pathway and
the regulation of classic E2F target genes are separable.
The above question is partially addressed by preliminary studies showing that the
deletion of a single allele of p53 rescues the cell cycle re-entry defect in E2f3-deficient
MEFs (data not shown). Lowering of p53 activity rather than its complete elimination
may indeed be sufficient to restore normal cell cycle re-entry. E2F target gene
expression will need to be examined in these cells. This experiment may demonstrate
that the primary function of E2F3 during cell cycle re-entry is through the regulation of
p L9ARF and p53.
Classic E2F target gene regulation in cycling cells is a more complex issue. In
fact, it is not clear whether one would expect to see a phenotype in this setting. Classic
E2F target gene transcription does not fluctuate as dramatically in cycling cells as it does
during cell cycle re-entry. Consequently, the requirement for transcriptional activation
by E2F may not be as great. Large-scale analysis of the transcriptional programs of
pl9Arf knockout and E2f3;pl9Arf double knockout MEFs by microarray did not reveal
any significant changes in gene expression (data not shown). This may indicate that
E2F3 controls proliferation through mechanisms not involving p19ARF or classic E2F
target genes.
E2F3 control of proliferation through other targets
Since classic E2F target gene expression is not altered in cycling E2f3-deficient
MEFs, it is necessary to expand the role of E2F in its regulation of cellular proliferation.
162
In fact, the role of E2F has already been expanding. Techniques such as microarrays and
ChIP have allowed the discovery of novel pathways that are responsive to E2F (Ishida et
al., 2001; Kalma et al., 2001; Kel et al., 2001; Ma et al., 2002; Markey et al., 2002;
Muller et al., 2001; Polager et al., 2002; Ren et al., 2002; Stanelle et al., 2002; Vernell et
al., 2003; Wells et al., 2002). These genes may account for the proliferation defect in
cycling E2f3--deficient MEFs. However, due to the lack of evidence supporting a
transcriptional defect as the cause of the proliferation impairment, it is also prudent to
consider other mechanisms.
Origin recognition represents one way that E2F has been shown to regulate cell
cycle progression that is independent of transcription. In Drosophila melanogaster, Rbf,
dE2F and dDP were found to interact with DmORC (Bosco et al., 2001). Additionally,
two E2F binding sites are present next to a latent origin of replication in the Epstein-Barr
virus (EBV) (Maser et al., 2001). A replication initiation site also exists in proximity to
the c-myc locus and its E2F binding sites. These findings suggest that some of the E2F
binding sites in the genome are used to direct sites of replication initiation. If this were
true, E2f3 loss could adversely affect replication by decreasing the number of available
origins.
E2f3-loss may also affect cell cycle progression outside of S-phase. This is
supported by the observation that cell cycle phasing appears to be relatively unchanged in
E2f3-deficient cells, despite their proliferation impairment. To verify that this is true, the
timing of mitotic events can be monitored using live imaging microscopy. DNA
condensation and segregation can be followed in live cells stably infected with histone
protein tagged with green fluorescent protein (GFP). This method has been used by
163
others to determine the relative length of mitosis in different circumstances. Rb knockout
MEFs were found to have a delay in mitosis using this technique (Hernando et al., 2004).
DISSECTING E2F3 FUNCTION
The E2F3 locus encodes two gene products, E2F3A and E2F3B, meaning that
phenotypes attributed to E2F3 in this dissertation could reflect the function of only
E2F3A, only E2F3B, or both genes (Lees et al., 1993; Leone et al., 2000). Another
possibility is that these gene products have additional functions, but act in opposition,
meaning that these functions are not apparent when both are deleted.
Thus far, distinguishing E2F3A function from E2F3B function has been difficult.
The fact that both proteins share a majority of amino acid sequence means that finding
specific antibodies to E2F3B is unlikely. Attempts aimed at the development of mouse
siRNA vectors specific for either E2F3A or E2F3B have been unsuccessful, meaning
these questions cannot currently be addressed. However, germline deletion specific for
either E2f3a or E2f3b are underway and hopefully this will permit us to distinguish the
shared and specific functions of the two E2F3 gene products. In the meantime, it is
possible to speculate on this topic based upon initial characterizations of E2F3A and
E2F3B.
Transcriptional activation by E2F3A
E2F3A has been extensively characterized as an activator of E2F target gene
expression (Lees et al., 1993). The fact E2f3-loss impairs cell cycle re-entry,
proliferation in cycling cells, and tumorigenesis is consistent with the function attributed
to E2F3A (Humbert et al., 2000; Ziebold et al., 2003; Ziebold et al., 2001).
164
E2F3A may have an important role during cell cycle re-entry. In quiescent cells
that have been stimulated to re-enter the cell cycle, E2F target genes must proceed from a
state of repression to activation, which could involve E2F3A. However, the rescue of
this phenotype in E2f3,pl9Arf and E2f3,p53 compound mutant MEFs at least means that
E2F3A is not essential for complete induction of E2F target gene expression during cell
cycle re-entry. E2f3a knockout MEFs could be examined to determine if the defect in
cell cycle re-entry and in the induction of E2F target gene expression is seperable from
the regulation of p19ARF/p53.
It may also be possible to establish a role for E2F3A in
the regulation of E2F target gene induction through the use of an E2F3A specific
antibody in ChIP assays. Even if there is no phenotype in the E2f3a knockout MEFs,
ChIP data may demonstrate that E2F3A participates with other activating E2Fs to
regulate E2F target gene expression in this context.
Since the mechanism behind the proliferation defect in cycling E2f3-deficient
MEFs is still unknown, the involvement of E2F3A in this process is an open question.
The function of E2F3A as an activator of E2F target gene expression has led to the
assumption that it would be important in cycling cells. Similar arguments have been
made in regard to the role of E2F3 in tumorigenesis.
It is tempting to attribute the
function of E2F3 in tumors to the control of proliferation through cell cycle regulation.
However, this is yet to be established and the differences between p19Arf and p53 null
tumors suggest that a more complex role is involved. Hopefully these issues can be
addressed with the aid of primary as well as transformed E2f3a-deficient MEFs.
165
Transcriptional repression by E2F3B
The function of E2F3B has not been definitively established, although E2F3B is
generally thought of as a repressor of E2F target gene expression (Leone et al., 2000). Its
expression pattern mirrors that of E2F4 and E2F5, the other repressive E2Fs. Most
notably, E2F3B is expressed in quiescent cells, where it is found in complex with pRb,
when E2F target gene expression is repressed. E2F3B also lacks the long amino terminus
found in E2F1, E2F2, and E2F3A, although no function has been assigned to this region.
Finally, its association with the p19Arf promoter and the phenotype of E2f3 knockout
MEFs is most consistent with E2F3B functioning as a repressor.
E2F3B may specifically repress the expression of additional genes besides
p19Arf, representing an important new class of E2F target genes. Repression of pl9Arf
by E2F3B has been clearly established during quiescence, and data for E2f3 knockout
MEFs suggests this is when E2F3B is essential for proper p9Arf regulation.
This could
be confirmed using E2f3b knockout MEFs. They would also be useful to confirm that
E2F3B continues to occupy the p]9Arf promoter during the cell cycle, although
deregulated gene expression is not observed in cycling cells. This experiment is
necessary because the ChIP assay requires an antibody that recognizes both E2F3A and
E2F3B, and therefore it is not possible to rule out the possibility that E2F3A participates
in p]9Arf regulation using this method in wild-type cells. Finally, the generation of
E2f3b knockout MEFs will also allow us to determine whether deregulation of
p19ARF/p53 is sufficient to cause a cell cycle re-entry defect.
Since the nature of the proliferation defect in cycling E2f3-deficient MEFs has not
been established, it is possible that repression by E2F3B may be important for cellular
166
proliferation. This question could be addressed using E2f3b-deficient cells. For this
purpose, it will also be useful to establish the subset of E2F target genes regulated by
E2F3B in cycling cells. This could be accomplished using ChIP microarrays on E2J3a
knockout MEFs. Similar studies could identify E2F3A target genes using E2f3b
knockout MEFs.
E2F3B could also have a key role in tumorigenesis, and E2f3b-deficient mice will
be useful to address this question. One of the disappointments of the E2f3 knockout
mouse is that it does not develop spontaneous tumors as the E2fl knockout mouse does
(Yamasaki et al., 1996). However, tumor onset in the E2fl knockout mouse is very late
and the proliferation impairment associated with E2f3-loss may obscure any tumor
development.
If regulation of proliferation is specifically attributable to E2F3A, it may
be possible to uncover a tumor suppressor phenotype in E2f3b knockout mice. It would
also be informative to generate E2f3b;E2f4 compound mutant mice, which should reduce
repression of E2F target gene expression mediated by pRb, and may also uncover a tumor
phenotype.
Transcriptional activation by E2F3B
Despite being grouped with the repressive E2Fs, E2F3B has properties that
suggest it could have dual roles as both an activator and repressor of E2F target gene
expression. This would make E2F3B unique among the E2Fs, as all other E2Fs have
been characterized as exclusively activators or repressors by most studies. The original
experiments characterizing the function of E2F3A were initially performed using an
amino terminal mutant lacking the residues encoded by the first exon (Lees et al., 1993).
This E2F3A mutant resembles E2F3B and behaves as a transcriptional activator in
167
reporter assays. This clearly establishes that E2F3B should have the potential to function
as an activator of E2F target gene expression under the correct circumstances. This also
contrasts with the repressive E2Fs, which function poorly in reporter assays.
The localization pattern of E2F3B makes one strong argument for its role as an
activator of E2F target gene expression.
E2F3B, like the activating E2Fs, possesses a
nuclear localization signal (in fact it is identical to that of E2F3A). Localization studies
place it predominantly in the nucleus (data not shown). In contrast, the repressive E2Fs
are primarily cytoplasmic due to nuclear export signals (Gaubatz et al., 2001; Verona et
al., 1997). They are only found in the nucleus when bound to pocket proteins, which hide
the NES. Once E2F4 and E2F5 are released from pocket protein association, they are
rapidly exported into the cytoplasm. E2F3B, on the other hand, remains in the nucleus at
this point, putting it at least in the right cellular compartment to activate E2F target gene
expression.
The possibility that E2F3B functions as an activator of E2F target gene expression
suggests an intriguing model. E2F1, and probably E2F2 and E2F3A, are destroyed by
the proteasome at the end of S-phase meaning these proteins have to be made anew at the
subsequent G 1/S transition. If activation of E2F target gene expression is required at this
stage, and not just relief of E2F mediated repression, then there is a potential problem.
This situation would also be faced during cell cycle re-entry, because the activating E2Fs
are not expressed in quiescent cells. However, E2F3B would be expressed and would be
in the nucleus. In this way, E2F3B could jump-start the G1/S transition and the
expression of classic E2F target genes.
168
CONCLUDING REMARKS
The studies in this dissertation were designed to understand the mechanism of
E2F3 function in cell cycle re-entry, asynchronous proliferation and tumorigenesis, with
a special focus on the roles played by p19ARF and p53 in these processes. As described
in detail above, E2F3 clearly regulates cellular proliferation through multiple
mechanisms. During cell cycle re-entry, pl9ARF-dependent
most critical. However, in cycling cells, pl9ARF-independent
mechanisms appear to be
mechanisms appear to be
the critical to the function of E2F3.
A complicating factor in studying the crosstalk between E2F and p19ARF/p53 is
that E2F appears to function both upstream and downstream of p19ARF/p53. As
indicated in the above sections, E2F is capable of regulating both p19ARF expression and
p53 activity to affect cellular proliferation. However, p19ARF and p53 also regulate the
cell cycle and modulate E2F activity, in part through p21CIP. It is important to keep this
in mind when interpreting these and future experiments.
These studies also begin to address some of the broad questions regarding E2F
function. E2F3 appears to be dispensable for the normal expression of many E2F target
genes in several settings. Although compensation by other E2Fs may be responsible for
this observation, cell and mouse system similar to those used in this dissertation may be
useful to test the models of E2F target gene specificity versus E2F dosage in the
regulation of E2F target genes. This question can be addressed through a combination of
experiments characterizing E2F target gene transcript levels by Northern or quantitative
real-time PCR as well as identification of E2F family members present at the promoters
of these target genes by ChIP. By performing this analysis in various E2f mutant
169
settings, one can begin to assemble a picture of how E2F target gene regulation is
accomplished.
Additionally, similar studies can address the question of whether E2F target gene
regulation occurs through activation or repression. For these studies, it may be useful to
include cells deficient for eitherpl9Arforp53.
These cells display increased expression
of E2F target genes. Again, experiments can be performed characterizing E2F target
gene transcript levels by Northern or quantitative real-time PCR as well as identification
of E2F family members present at the promoters of these target genes. For those E2F
target genes whose expression is altered by disruption of the p53 pathway, one can ask
whether it is due to a reduction of repressive E2Fs or an increase of activating E2Fs at the
promoter by Ch[P. It may also be useful to employ E2f;pl9Arf and/or E2f;p53
compound mutant MEFs in these studies. Through understanding of these different
mechanisms E2F function and target gene regulation, we will be better able to understand
the important of the deregulation of E2F activity in cancers.
170
References.
Ameyar-Zazoua, M., Wisniewska, M. B., Bakiri, L., Wagner, E. F., Yaniv, M., and
Weitzman, J. B. (2005). AP-1 dimers regulate transcription of the p14/p19ARF tumor
suppressor gene. Oncogene 24, 2298-2306.
Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden,
K. H. (1998). p14ARF links the tumour suppressors RB and p53. Nature 395, 124-125.
Berthet, C., Aleem, E., Coppola, V., Tessarollo, L., and Kaldis, P. (2003). Cdk2 knockout
mice are viable. Curr Biol 13, 1775-1785.
Bornstein, G., Bloom, J., Sitry-Shevah, D., Nakayama, K., Pagano, M., and Hershko, A.
(2003). Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cipl in S phase. J
Biol Chem 278, 25752-25757.
Bosco, G., Du, W., and Orr-Weaver, T. L. (2001). DNA replication control through
interaction of E2F-RB and the origin recognition complex. Nat Cell Biol 3, 289-295.
Brugarolas, J., Chandrasekaran, C., Gordon, J. I., Beach, D., Jacks, T., and Hannon, G. J.
(1995). Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377,
552-557.
Brummelkamp, T. R., Kortlever, R. M., Lingbeek, M., Trettel, F., MacDonald, M. E., van
Lohuizen, M., and Bernards, R. (2002). TBX-3, the gene mutated in Ulnar-Mammary
Syndrome, is a negative regulator of p19ARF and inhibits senescence. J Biol Chem 277,
6567-6572.
Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J. P., Sedivy, J. M.,
Kinzler, K. W., and Vogelstein, B. (1998). Requirement for p53 and p21 to sustain G2
arrest after DNA damage. Science 282, 1497-1501.
de Stanchina, E., McCurrach, M. E., Zindy, F., Shieh, S. Y., Ferbeyre, G., Samuelson, A.
V., Prives, C., Roussel, M. F., Sherr, C. J., and Lowe, S. W. (1998). E1A signaling to p53
involves the p19(ARF) tumor suppressor. Genes Dev 12, 2434-2442.
Deng, C., Zhang, P., Harper, J. W., Elledge, S. J., and Leder, P. (1995). Mice lacking
p21CIP1/WAF1 undergo normal development, but are defective in GI checkpoint
control. Cell 82, 675-684.
Dimri, G. P., Itahana, K., Acosta, M., and Campisi, J. (2000). Regulation of a senescence
checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor.
Mol Cell Biol 20, 273-285.
el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin,
D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993). WAFI, a potential
mediator of p53 tumor suppression. Cell 75, 817-825.
171
Fridman, J. S., and Lowe, S. W. (2003). Control of apoptosis by p53. Oncogene 22,
9030-9040.
Gaubatz, S., Lees, J. A., Lindeman, G. J., and Livingston, D. M. (2001). E2F4 is exported
from the nucleus in a CRM1-dependent manner. Mol Cell Biol 21, 1384-1392.
Geng, Y., Yu, Q., Sicinska, E., Das, M., Schneider, J. E., Bhattacharya, S., Rideout, W.
M., Bronson, R. T., Gardner, H., and Sicinski, P. (2003). Cyclin E ablation in the mouse.
Cell 114, 431-443.
Giangrande, P. H., Zhu, W., Rempel, R. E., Laakso, N., and Nevins, J. R. (2004).
Combinatorial gene control involving E2F and E Box family members. Embo J 23, 13361347.
Hernando, E., Nahle, Z., Juan, G., Diaz-Rodriguez, E., Alaminos, M., Hemann, M.,
Michel, L., Mittal, V., Gerald, W., Benezra, R., et al. (2004). Rb inactivation promotes
genomic instability by uncoupling cell cycle progression from mitotic control. Nature
430, 797-802.
Hiyama, H., Iavarone, A., and Reeves, S. A. (1998). Regulation of the cdk inhibitor p21
gene during cell cycle progression is under the control of the transcription factor E2F.
Oncogene 16, 1513-1523.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
Iliakis, G., Wang, Y., Guan, J., and Wang, H. (2003). DNA damage checkpoint control in
cells exposed to ionizing radiation. Oncogene 22, 5834-5847.
Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M., and Nevins, J. R.
(2001). Role for E2F in control of both DNA replication and mitotic functions as
revealed from DNA microarray analysis. Mol Cell Biol 21, 4684-4699.
Itahana, K., Dimri, G. P., Hara, E., Itahana, Y., Zou, Y., Desprez, P. Y., and Campisi, J.
(2002). A role for p53 in maintaining and establishing the quiescence growth arrest in
human cells..J Biol Chem 277, 18206-18214.
Jacobs, J. J., Keblusek, P., Robanus-Maandag, E., Kristel, P., Lingbeek, M., Nederlof, P.
M., van Welsem, T., van de Vijver, M. J., Koh, E. Y., Daley, G. Q., and van Lohuizen,
M. (2000). Senescence bypass screen identifies TBX2, which represses Cdkn2a
(p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet 26, 291-299.
Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A., and van Lohuizen, M. (1999).
The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and
senescence through the ink4a locus. Nature 397, 164-168.
172
Kalma, Y., Marash, L., Lamed, Y., and Ginsberg, D. (2001). Expression analysis using
DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication
genes including replication protein A2. Oncogene 20, 1379-1387.
Kamijo, T., van de Kamp, E., Chong, M. J., Zindy, F., Diehl, J. A., Sherr, C. J., and
McKinnon, P. J. (1999). Loss of the ARF tumor suppressor reverses premature
replicative arrest but not radiation hypersensitivity arising from disabled atm function.
Cancer Res 59, 2464-2469.
Kel, A. E., Kel-Margoulis, O. V., Farnham, P. J., Bartley, S. M., Wingender, E., and
Zhang, M. Q. (2001). Computer-assisted identification of cell cycle-related genes: new
targets for E2F transcription factors. J Mol Biol 309, 99-120.
Kozar, K., Ciemerych, M. A., Rebel, V. I., Shigematsu, H., Zagozdzon, A., Sicinska, E.,
Geng, Y., Yu, Q., Bhattacharya, S., Bronson, R. T., et al. (2004). Mouse development
and cell proliferation in the absence of D-cyclins. Cell 118, 477-491.
Lees, J. A., Saito, M., Vidal, M., Valentine, M., Look, T., Harlow, E., Dyson, N., and
Helin, K. (1993). The retinoblastoma protein binds to a family of E2F transcription
factors. Mol Cell Biol 13, 7813-7825.
Leone, G., Nuckolls, F., Ishida, S., Adams, M., Sears, R., Jakoi, L., Miron, A., and
Nevins, J. R. (2000). Identification of a novel E2F3 product suggests a mechanism for
determining specificity of repression by Rb proteins. Mol Cell Biol 20, 3626-3632.
Lingbeek, M. E., Jacobs, J. J., and van Lohuizen, M. (2002). The T-box repressors TBX2
and TBX3 specifically regulate the tumor suppressor gene p14ARF via a variant T-site in
the initiator. J Biol Chem 277, 26120-26127.
Ma, Y., Croxton, R., Moorer, R. L., Jr., and Cress, W. D. (2002). Identification of novel
E2F1-regulated genes by microarray. Arch Biochem Biophys 399, 212-224.
Maeda, T., Hobbs, R. M., Merghoub, T., Guernah, I., Zelent, A., Cordon-Cardo, C.,
Teruya-Feldstein, J., and Pandolfi, P. P. (2005). Role of the proto-oncogene Pokemon in
cellular transformation and ARF repression. Nature 433, 278-285.
Malumbres, M., Sotillo, R., Santamaria, D., Galan, J., Cerezo, A., Ortega, S., Dubus, P.,
and Barbacid, M. (2004). Mammalian cells cycle without the D-type cyclin-dependent
kinases Cdk4 and Cdk6. Cell 118, 493-504.
Markey, M. P., Angus, S. P., Strobeck, M. W., Williams, S. L., Gunawardena, R. W.,
Aronow, B. J., and Knudsen, E. S. (2002). Unbiased analysis of RB-mediated
transcriptional repression identifies novel targets and distinctions from E2F action.
Cancer Res 62, 6587-6597.
Maser, R. S., Mirzoeva, O. K., Wells, J., Olivares, H., Williams, B. R., Zinkel, R. A.,
Farnham, P. J., and Petrini, J. H. (2001). Mrel 1 complex and DNA replication: linkage to
E2F and sites of DNA synthesis. Mol Cell Biol 21, 6006-6016.
173
Molofsky, A. V., He, S., Bydon, M., Morrison, S. J., and Pardal, R. (2005). Bmi-1
promotes neural stem cell self-renewal and neural development but not mouse growth
and survival by repressing the pl6Ink4a and pl9Arf senescence pathways. Genes Dev 19,
1432-1437.
Muller, H., Bracken, A. P., Vernell, R., Moroni, M. C., Christians, F., Grassilli, E.,
Prosperini, E., Vigo, E., Oliner, J. D., and Helin, K. (2001). E2Fs regulate the expression
of genes involved in differentiation, development, proliferation, and apoptosis. Genes
Dev 15, 267-285.
Niculescu, A. B., 3rd, Chen, X., Smeets, M., Hengst, L., Prives, C., and Reed, S. I.
(1998). Effects of p21(Cipl/Wafl) at both the G1/S and the G2/M cell cycle transitions:
pRb is a critical determinant in blocking DNA replication and in preventing
endoreduplication. Mol Cell Biol 18, 629-643.
Ortega, S., Prieto, I., Odajima, J., Martin, A., Dubus, P., Sotillo, R., Barbero, J. L.,
Malumbres, M., and Barbacid, M. (2003). Cyclin-dependent kinase 2 is essential for
meiosis but not for mitotic cell division in mice. Nat Genet 35, 25-31.
Palmero, I., Pantoja, C., and Serrano, M. (1998). p19ARF links the tumour suppressor
p53 to Ras. Nature 395, 125-126.
Parisi, T., Beck, A. R., Rougier, N., McNeil, T., Lucian, L., Werb, Z., and Amati, B.
(2003). Cyclins El and E2 are required for endoreplication in placental trophoblast giant
cells. Embo J 22, 4794-4803.
Park, I. K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L., Morrison, S.
J., and Clarke, M. F. (2003). Bmi-1 is required for maintenance of adult self-renewing
haematopoietic stem cells. Nature 423, 302-305.
Polager, S., Kalma, Y., Berkovich, E., and Ginsberg, D. (2002). E2Fs up-regulate
expression of genes involved in DNA replication, DNA repair and mitosis. Oncogene 21,
437-446.
Radhakrishnan, S. K., Feliciano, C. S., Najmabadi, F., Haegebarth, A., Kandel, E. S.,
Tyner, A. L., and Gartel, A. L. (2004). Constitutive expression of E2F-1 leads to p21dependent cell cycle arrest in S phase of the cell cycle. Oncogene 23, 4173-4176.
Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A., and Dynlacht,
B. D. (2002). E2F integrates cell cycle progression with DNA repair, replication, and
G(2)/M checkpoints. Genes Dev 16, 245-256.
Rowland, B. D., Denissov, S. G., Douma, S., Stunnenberg, H. G., Bernards, R., and
Peeper, D. S. (2002). E2F transcriptional repressor complexes are critical downstream
targets of pl9(ARF)/p53-induced proliferative arrest. Cancer Cell 2, 55-65.
Sherr, C. J., and Roberts, J. M. (1999). CDK inhibitors: positive and negative regulators
of GI-phase progression. Genes Dev 13, 1501-1512.
174
Stanelle, J., Stiewe, T., Theseling, C. C., Peter, M., and Putzer, B. M. (2002). Gene
expression changes in response to E2F1 activation. Nucleic Acids Res 30, 1859-1867.
Taylor, W. R., and Stark, G. R. (2001). Regulation of the G2/M transition by p53.
Oncogene 20, 1803-1815.
Vernell, R., Helin, K., and Muller, H. (2003). Identification of target genes of the
p16INK4A-pRB-E2F pathway. J Biol Chem 278, 46124-46137.
Verona, R., Moberg, K., Estes, S., Starz, M., Vernon, J. P., and Lees, J. A. (1997). E2F
activity is regulated by cell cycle-dependent changes in subcellular localization. Mol Cell
Biol 17, 7268-7282.
Weitzman, J. B., Fiette, L., Matsuo, K., and Yaniv, M. (2000). JunD protects cells from
p53-dependent senescence and apoptosis. Mol Cell 6, 1109-1119.
Wells, J., Graveel, C. R., Bartley, S. M., Madore, S. J., and Farnham, P. J. (2002). The
identification of E2F1-specific target genes. Proc Natl Acad Sci U S A 99, 3890-3895.
Xu, Y., Yang, E. M., Brugarolas, J., Jacks, T., and Baltimore, D. (1998). Involvement of
p53 and p21 in cellular defects and tumorigenesis in Atm-/- mice. Mol Cell Biol 18,
4385-4390.
Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996).
Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85, 537-548.
Ziebold, U., Lee, E. Y., Bronson, R. T., and Lees, J. A. (2003). E2F3 loss has opposing
effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but
metastasis of medullary thyroid carcinomas. Mol Cell Biol 23, 6542-6552.
Ziebold, U., Reza, T., Caron, A., and Lees, J. A. (2001). E2F3 contributes both to the
inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev
15, 386-391.
Zindy, F., Eischen, C. M., Randle, D. H., Kamijo, T., Cleveland, J. L., Sherr, C. J., and
Roussel, M. F. (1998). Myc signaling via the ARF tumor suppressor regulates p53dependent apoptosis and immortalization. Genes Dev 12, 2424-2433.
175
Appendix A
E2J3-Ioss leads to increased cell size
Aaron Aslanian and Jacqueline A. Lees
Author's contributions: Figure 1, 2 and 3
176
Cell growth is necessary prior to cell division to maintain a consistent range in
cell size. In yeast, cell growth is monitored to ensure that a minimize cell size is
achieved prior to cell division(Kellogg, 2003; Rupes, 2002). However, it is not clear
whether mammalian cells also regulate cell growth, or whether cell growth is merely a
reflection of the rate at which cell division occurs(Coelho and Leevers, 2000; Jorgensen
and Tyers, 2004). Several models have been proposed, but so far the available data does
not support a single, unifying paradigm.
Cell growth and cell division play a significant role in determining the final size
of an adult organism. Both intracellular and extracellular signals govern size
determination and interestingly these same signals can influence aging and cancer(Liang
et al., 2003). Within an organism, cells attain a variety of shapes and sizes in accordance
with their intended functions. However, developmental regulation of cell size may
involve more complex interactions than merely cell growth regulation, so one should be
cautious when evaluating conclusions made in different systems.
Several pathways have been implicated in the regulation of cell growth. The
mTOR pathway regulates cell growth by monitoring nutrient levels(Fingar et al., 2002;
Tee and Blenis, 2005). Insulin-like growth factor (IGF) signaling regulates many aspects
of both cell growth and cell division(Dupont et al., 2003). Both these pathways
coordinate protein translation within the cell, in particular through the regulation of
eIF4e. Cell cycle status can also affect cell size. Senescent cells are characteristically
large despite being arrested in G1.
The transcription factor c-myc has been implicated in the regulation of both cell
division and cell growth. The initial characterization of c-myc deficient cells revealed a
177
significant delay in progression through the two gap phases of the cell cycle(Mateyak et
al., 1997). Overall cycle progression is drastically delayed in c-myc deficient cells, while
cell size is unaffected. More recent studies have shown that c-Myc regulates several
proteins involved in ribosome biogenesis and protein translation, two key processes that
impinge on cell growth(Schmidt, 1999; Schmidt, 2004).
The E2F/Rb pathway has also be shown to module cell growth, although it is not
yet clear whether this represents a function that can be separated from the regulation of
cellular proliferation. Rb-deficient MEFs are slightly increased in cell size, and this
phenotype is reversed by the additional deletion of p21Cip(Brugarolas et al., 1998).
MEFs lacking all three pocket proteins show a much more dramatic increase in cell
size(Sage et al., 2000). Mice expressing an Rb transgene are dwarfs, and this has been
attributing to elevated IGF levels found in the plasma of these mice(Bignon et al., 1993;
Nikitin et al., 2001). These data support a role for pRb in growth control. Finally, both
E2f3 and E2f4 knockout mice are severely runted, although the cause of this phenotype is
still unknown(Humbert et al., 2000a; Humbert et al., 2000b).
We observed that E2f3-deficient MEFs appear larger in size than wild-type MEFs
(Figure 1A). To verify that E2f3 knockout MEFs are larger than wild-type MEFs, we
incubated them with 5-chloromethylfluorescein diacetate (CMFDA), a cell membrane
permeable dye that is converted into a membrane-impermeable
cell (Figure
form once inside in the
B). This allowed cell size and cell shape to be measured by
immunofluorescent microscopy. Quantification of mean cell area demonstrated that E2f3
knockout MEFs are 18.8 percent larger than wild-type MEFs (Figure IC). No change in
cell shape was observed.
178
Figure 1.
A)
E2f3 knockout
wildtype
B)
C)
3000
--4000 -
I
3500-
r.
--- _-
2500
4-
_
-----
20001500
1000
500
0-
-
wildtype
_
E2f3 knockout
--
-
Figure 1. The loss of E2f3 results in an increase in cell size. (A) Phase contrast images of
representative wildtype and E2f3 knockout MEFs. (B) Immunofluorsence images of MEFs
labeled with 5-chloromethylfluorescein
diacetate [CMFDA] (green), Hoescht (blue) and
merged. (C) Cellomics quantification of three wildtype and three E2f3 knockout MEF lines,
each in triplicate. Each data point represents the mean area of a minimum of 100 cells.
To further characterize the effect of E2f3-loss on cell size, we analyzed wild-type
and E2f3 knockout MEFs by FACS. Again, a 14.8 percent increase in mean cell size was
observed in E2f3 knockout MEFs relative to wild-type MEFs (Figure 2A,B). Changes in
cell cycle distribution can affect the mean cell size in a population, because G2/M cells
are larger than G1 cells. Using relative DNA content, we compared cell size specifically
in populations of either GI or G2/M cells. The increase in cell size was independent of
cell cycle phase, showing a 14.3 percent increase in cell size among G1 cells and a 16.2
percent increase in cell size among G2/M cells (Figure 2C,D). This is consistent with our
previous observation that the loss of E2f3 does not significantly alter the distribution of
cells within the cell cycle. This method may also indicate that the difference in cell size
we observed is not merely a reflection of differences in cell spreading or motility.
The genetic requirements for E2f3 status to modulate cell size were examined by
FACS, using compound mutant MEFs. The loss or either pl9Arf or p53 results in an
increase in proliferation as well as a slight decrease in cell size (data not shown).
E2f3;pl9Arf double knockout MEFs were 12.6 percent larger than pl 9Arf knockout
MEFs (Figure 3A). Similarly, E2f3;p53 double knockout MEFs were 8.5 percent larger
than p53 knockout MEFs (Figure 3B). So as with the regulation of cellular proliferation,
E2f3 status modulates cell size independent of p19ARF/p53. p6INK4A
is a key
upstream regulator of the Rb pathway whose expression correlates with the increase in
cell size of senescent cells. However, E2f3;Ink4a double knockout MEFs were 13.2
percent larger than Ink4A knockout MEFs, demonstrating that p16INK4A is also not
required for this phenotype (Figure 3C). Additionally, the loss of E2f3 does not appear to
affect cellular senescence.
180
Figure 2.
A)
B)' 11
1./.
n
I
N
1 -I
(A
= 0.8 ' 0.6.
0.4) 0.2-
0-
FSC-H
I
wildtype
C)
wildtype
D
>
Qw~~~~~.s
E
FSC2H
D)
t
Iq
C
E2f3
knockout
E2f3 knockout
Ii
_t
t 0n-..41.-X*.
V8 B.S
l
-
e
.
1
4M
. . W.1I.~.
.
-
O
,iI
So
IM
1.2
N
1
0.8
eO
0.6
C.
0.4
ca
* wildtype
O E2f3 knockout
C).2
0
G1 cells
G2/M cells
Figure 2. Increased cell size due to E2f3-loss is cell cycle independent.
(A) FACS histogram of
forward scatter (FSC) versus counts for wildtype (blue line) and E2f3 knockout MEFs (red line).
(B) Bar graph comparing mean FSC of four wildtype and four E2f3 knockout MEFs. (C) FACS
plot of FSC versus relative DNA content measured by propidium iodide (PI) staining for
wildtype and E2f3 knockout MEFs. (D) Bar graph comparing mean FSC gated for 2N (G1
cells) and 4N DNA content (G2/M cells) for wildtype (black bars) and E2f3 knockout MEFs
(white bars).
Figure 3.
A)
0ir)
1.2 -
1,,
1 -
*C
_
N
0.8
0.6
0.4
0.2
0
C
w
-
I·
1.
-L
V.
1 -
0.8 -
o 0.6A
>
.-
4
-
w 0.2-
c
0-
tu
w
G1 cells
Cl
B)
w 1.2 !
1.2
.
w
C.)
N
1
---
0.8
rA
c
0.6
00U:
.> 0.4
=t
0.2
.6-a
1
0.8
0.6
0.4
Ct
0
---
I
I A\
sv
V)oO
0.2
0
O
3
G1 cells
14
C)
w
*
,
G2/M
cells
N
*-
Im
I
1.2-
1.2
1 -
0.8
0.6
(D
r-Cj
0.4
ct 0.2
G2/M
cells
N
-
1
0.8
w
;-4
I
.
C)
tO
0.6
0;.
0.4
om.
w~
+
-
'rl'
0=
O
0.2
0
G1 cells G2/M
14
NL
cells
Figure 3. continued
Figure 3. Increased cell size due to E2f3 loss is independent of pl9Arf/p53.
(A) Bar
graph comparing mean FSC of four pl 9Arf knockout and four E2f3,pl 9Arf double
knockout MEFs, left. Bar graph comparing mean FSC gated for 2N (GI cells) and 4N
DNA content (G2/M cells) for p19Arf knockout (black bars) and E2f3;pl9Arf double
knockout MEFs (white bars), right. (B) Bar graph comparing mean FSC of four p53
knockout and four E2f3;p53 double knockout MEFs, left. Bar graph comparing mean
FSC gated for 2N (G1 cells) and 4N DNA content (G2/M cells) for p53 knockout
(black bars) and E2f3;p53 double knockout MEFs (white bars), right. (C) Bar graph
comparing mean FSC of four Ink4a knockout and four E2f3;Ink4a double knockout
MEFs, left. Bar graph comparing mean FSC gated for 2N (G 1 cells) and 4N DNA
content (G2/M cells) for Ink4a knockout (black bars) and E2f3;Ink4a double knockout
MEFs (white bars), right.
The currently available data would indicate that the observed increase in cell size
of E2f3-deficient MEFs is a direct result of a general slowing of cell cycle progression.
However, there are still several reasons why this phenotype should not be dismissed.
First is the role of c-myc as a regulator of cell growth. Given the similarities between cmyc function and that of E2F, there may be a role of E2F in growth regulation that is
independent of its control of cell cycle progression. It will be helpful to examine whether
components of the mTOR and IGF signaling pathways are directly regulated by E2F, and
how they might be affected in E2f3-deficient MEFs. Second, the loss of E2f3 does not
produce significant changes in gene expression, meaning that other processes such as
translation may be responsible for the proliferation defect. Given the connection between
translation and cell growth control, it is possible that impaired translation is responsible
for causing a slowing of cell cycle in E2J3-deficient MEFs. Finally, the role of pRb in
IGF signaling in mice demands that we consider a role for E2F as well. Defects in IGF
signaling would be consistent with several of the cell and mouse phenotypes associated
with E2f3 loss.
184
References.
Bignon, Y. J., Chen, Y., Chang, C. Y., Riley, D. J., Windle, J. J., Mellon, P. L., and Lee,
W. H. (1993). Expression of a retinoblastoma transgene results in dwarf mice. Genes Dev
7, 1654-1662.
Brugarolas, J., Bronson, R. T., and Jacks, T. (1998). p2 1 is a critical CDK2 regulator
essential for proliferation control in Rb-deficient cells. J Cell Biol 141, 503-514.
Coelho, C. M., and Leevers, S. J. (2000). Do growth and cell division rates determine cell
size in multicellular organisms? J Cell Sci 113 ( Pt 17), 2927-2934.
Dupont, J., Pierre, A., Froment, P., and Moreau, C. (2003). The insulin-like growth factor
axis in cell cycle progression. Horm Metab Res 35, 740-750.
Fingar, D. C., Salama, S., Tsou, C., Harlow, E., and Blenis, J. (2002). Mammalian cell
size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes
Dev 16, 1472-1487.
Humbert, P. O., Rogers, C., Ganiatsas, S., Landsberg, R. L., Trimarchi, J. M., Dandapani,
S., Brugnara, C., Erdman, S., Schrenzel, M., Bronson, R. T., and Lees, J. A. (2000a).
E2F4 is essential for normal erythrocyte maturation and neonatal viability. Mol Cell 6,
281-291.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000b). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
Jorgensen, P., and Tyers, M. (2004). How cells coordinate growth and division. Curr Biol
14, R1014-1027.
Kellogg, D. R. (2003). Weel-dependent mechanisms required for coordination of cell
growth and cell division. J Cell Sci 116, 4883-4890.
Liang, H., Masoro, E. J., Nelson, J. F., Strong, R., McMahan, C. A., and Richardson, A.
(2003). Genetic mouse models of extended lifespan. Exp Gerontol 38, 1353-1364.
Mateyak, M. K., Obaya, A. J., Adachi, S., and Sedivy, J. M. (1997). Phenotypes of c-
Myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell
Growth Differ 8, 1039-1048.
Nikitin, A., Shan, B., Flesken-Nikitin, A., Chang, K. H., and Lee, W. H. (2001). The
retinoblastoma gene regulates somatic growth during mouse development. Cancer Res
61, 3110-3118.
Rupes, I. (2002). Checking cell size in yeast. Trends Genet 18, 479-485.
185
Sage, J., Mulligan, G. J., Attardi, L. D., Miller, A., Chen, S., Williams, B., Theodorou, E.,
and Jacks, T. (2000). Targeted disruption of the three Rb-related genes leads to loss of
G(1) control and immortalization. Genes Dev 14, 3037-3050.
Schmidt, E. V. (1999). The role of c-myc in cellular growth control. Oncogene 18, 29882996.
Schmidt, E. V. (2004). The role of c-myc in regulation of translation initiation. Oncogene
23, 3217-3221.
Tee, A. R., and Blenis, J. (2005). mTOR, translational control and human disease. Semin
Cell Dev Biol 16, 29-37.
186
Appendix B
E23-loss impairs E2F target gene expression in a pure strain background
Aaron Aslanian and Jacqueline A. Lees
Author's contributions: Figure 1
187
We and others have previously characterized the effect of E2f3-deficiency in
various settings. This has been done, when possible, in pure strain backgrounds
(typically 129Sv) as well as in a mixed strain background comprised of C57B16 and
129Sv. E2f3-deficient mice in any pure strain background are not viable, whereas in a
mixed strain background approximately a quarter to a third of the expected animals are
observed (Aslanian et al., 2004; Cloud et al., 2002; Humbert et al., 2000). Given that
multiple pure strain backgrounds yield lethality of E2f3 knockout mice, it seems likely
that hybrid vigor rather than a genetic modifier is responsible for this phenotype.
The loss of E2f3 results in a proliferation defect in mixed strain background
MEFs (Humbert et al., 2000). These cells also display an increase in cell size. However,
there is no change in E2F target gene expression in these cells relative to wild-type
MEFs. This was surprising because E2F3 was thought to be a key regulator of E2F target
gene expression, and fulfills this role during cell cycle re-entry (Aslanian et al., 2004;
Giangrande et al., 2004; Humbert et al., 2000; Leone et al., 1998). We characterized
E2f3 knockout MEFs in a pure 129Sv strain background to determine what effect strain
background has in this setting. As in a mixed strain background, pure 129Sv E2f3
knockout MEFs possess a proliferation defect (Figure 1A). They also show a 16.5
percent increase in cell size (Figure B,C). Interestingly, pure 129Sv E2f3 knockout
MEFs also exhibit decreased expression of E2F target genes (Figure D).
In general, E2f3-loss appears to have a more severe effect in pure strain
backgrounds compared to mixed strain backgrounds. Most importantly, we demonstrate
that the loss of E2f3 does impact E2F target gene expression in MEFs of a pure strain
background. These cells may be more sensitive to the loss of E2f3, although the reason
188
Figure 1.
A)
B)
oN 1.2
2500000
*
2000000
1
a) 0.8
0.6
E 1500000
·
-1000000
0.4
0.2
----
0
500000
U)
C14
0)
+-4
1
2
3
4
C)
:z
4M
-3 -0
C111
"-1
Cr4 o
. r--
days
o,
-1
11:
D)
1.2
N
1
0.8
.,in
Q) 0.6
'141k ... WO
4--
- t
0.4
Ct·
0.2
-Wm"
0
G1 cells
* wildtype
G2/M cells
'WllllW"Oftrll
actin
p107
cyclin A
cdc2
PCNA
p19ARF
p21CIP
0 E2f3 knockout
Figure 1. The loss of E2f3 results in reduced E2F target gene expression in MEFs derived from
a pure 129Sv strain background. (A) Pure 129Sv wildtype (black squares, solid line) and E2f3
knockout MEFs (white squares, dashed line) were initially plated at 3*10A5 per 6cm plate and
cell number was counted for a period of four days. (B) Bar graph comparing mean forward
scatter (FSC) of four pure 129Sv wildtype and four pure 129Sv E2f3 knockout MEFs. (C) Bar
graph for pure 129Sv wildtype and E2f3 knockout MEFs comparing mean FSC gated for 2N
(G1 cells) and 4N DNA content (G2/M cells) measured by propidium iodide (PI) staining. (D)
Representative western blotting of 10OOtgwhole cell extract from pure 129Sv wildtype and
E2f3 knockout MEFs for actin (sc-1616), p107 (sc-318), cyclin A (sc-596), cdc2 (sc-54),
PCNA (sc.-56), p19ARF (ab80-100), and p21CIP (sc-6246) protein levels.
for this is unclear. Hybrid vigor may be responsible for the absence of any change in E2F
target gene expression in mixed strain background E2f3-deficient MEFs. This data is
also consistent with observations that strain background can have a profound impact on
cancer-relevant phenotypes.
Experiments described earlier in this dissertation using E2f3;p53 compound
mutant MEFs were performed in a pure strain background (129Sv). The analysis of these
cells showed that E2f3-loss does not affect the levels of E2F target genes in p53 null cells
(Chapter 4). When considered with the above data, this suggests that p53-loss is capable
of suppressing the defect in E2F target gene expression observed in pure 129Sv strain
background MEFs. This parallels the ability of p19ARF/p53 loss to suppress defects in
E2F target gene expression during cell cycle re-entry (Aslanian et al., 2004). This also
suggests that the mechanism by which p19ARF/p53 suppress defects in E2F target gene
expression is still functional in cycling cells. This further indicates that the mechanism
behind the proliferation defect in cycling E2J3-deficient cells is mutually exclusive from
classic E2F target gene regulation and probably not operating during cell cycle re-entry.
190
References.
Aslanian, A., Iaquinta, P. J., Verona, R., and Lees, J. A. (2004). Repression of the Arf
tumor suppressor by E2F3 is required for normal cell cycle kinetics. Genes Dev 18, 14131422.
Cloud, J. E., Rogers, C., Reza, T. L., Ziebold, U., Stone, J. R., Picard, M. H., Caron, A.
M., Bronson, R. T., and Lees, J. A. (2002). Mutant mouse models reveal the relative roles
of E2F1 and E2F3 in vivo. Mol Cell Biol 22, 2663-2672.
Giangrande, P. H., Zhu, W., Rempel, R. E., Laakso, N., and Nevins, J. R. (2004).
Combinatorial gene control involving E2F and E Box family members. Embo J 23, 13361347.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
Leone, G., DeGregori, J., Yan, Z., Jakoi, L., Ishida, S., Williams, R. S., and Nevins, J. R.
(1998). E2F3 activity is regulated during the cell cycle and is required for the induction
of S phase. Genes Dev 12, 2120-2130.
191
Appendix C
The role of E2f3 in tumorigenesis in p53 heterozygous mice
Aaron Aslanian and Jacqueline A. Lees
Author's contributions: Figure 1 and 2; Table 1, 2 and 3
192
Rapid, spontaneous tumor development is responsible for the mortality of p53
knockout mice prior to ten months of age (Donehower et al., 1992; Harvey et al., 1993;
Jacks et al., 1994; Purdie et al., 1994). These mice primarily develop thymic lymphomas
and soft tissue sarcomas, with some variation in the frequency of these and other tumors
that is dependent on the strain background of the mice. p53 heterozygous mice are also
highly prone to spontaneous tumor development. However, tumor onset occurs much
later and a more diverse spectrum of tumors is observed.
Several studies have characterized tumorigenesis in p53 heterozygous mice.
Approximately half of these mice develop tumors by 18 months of age. A majority of
tumors observed in p53 heterozygous mice are soft tissue sarcomas. Lymphomas are also
very common and carcinomas are occasionally observed. Loss of heterozygosity (LOH)
is typical seen in most tumors, although p53 is commonly inactivated through mutation as
well. To this end, recent studies have analyzed survival and tumor spectrums in point
mutant p53 mice (Lang et al., 2004; Olive et al., 2004). In these mice there is some
change in tumor spectrum, but no overall change in survival relative to mice
heterozygous for the null allele of p53.
The difference in tumor spectrum between p53 knockout and p53 heterozygous
mice can be attributed to several factors. First, cells that give rise to tumors in p53
knockout mice begin their life without any p53, and subsequent mutations take place in
such an environment.
In contrast, cells that give rise to tumors in p53 heterozygous mice
probably accumulate several mutations prior to p53 inactivation. Second, the complete
absence of p53 in certain tissues may be sufficient to initiate rapid tumorigenesis, leading
to the development of tumors in these tissues before tumors in other tissues have time to
193
manifest themselves. In the p53 heterozygous mice, the likelihood of p53 inactivation is
approximately equal across tissues, meaning that no one type of tumor would have an
advantage. Third, tumors that arise in p53 knockout mice develop in an animal without
p53, and whatever gene expression program it regulates. Tumors that arise in p53
heterozygous mice are primarily in an environment of cells with functional p53 activity.
Surrounding stromal cells can profoundly influence tumor development.
We have shown that E2f3 status has no bearing on the survival of p53 knockout
mice, and does not appear to influence tumor spectrum. The above reasoning led us to
believe that an affect of E2f3 status may be more likely to reveal itself in p53
heterozygous mice. To this end, E2f3;p53 double heterozygous mice in a pure C57B16
strain background were intercrossed with E2f3;p53 double heterozygous mice in a pure
129Sv strain background. p53 heterozygous progeny were maintained and monitored to
assess the effect of E2f3 status on tumor development. Because of the reduced viability
of E2f3-deficient mice, these animals required a longer period of time to generate
sufficient numbers for this study. As a result, most p53 heterozygous mice that are also
E2f3 knockout are still alive and have yet to reach the window of tumor development
typically observed for p53 heterozygous mice. Consequently, only the effect of E2f3
heterozygosity will be discussed below.
E2f3 heterozygosity produced a slight effect on the survival of p53 heterozygous
mice (Figure 1). At eighteen months of age, survival of p53 heterozygous mice was at
42.3 percent, which is similar to what has previously been reported in other studies
(Table 1). The loss of a single allele of E2f3 boosted survival to 57.9 percent. The effect
of E2f3 status can also be observed through the age at which 50 percent mice are still
194
Figure 1.
1
l
T
0.9
OL
=4
0.8
..
.> 0.7
;>
0.6
v:
0.5
CQ
cl~
0.4
1
---
---oE2f3 heterozygous (n=38)
0.3
C).
E2f3 wildtype (n=26)
0.2
0. 1
0
!
I
0
100
200
3
4
300
400
II
500
I
iI
600
iI
700
days
Figure 1. E2f3 heterozygosity modestly extends the survival of p53 heterozygous
mice. p53 heterozygous mice that were either E2f3 wildtype (n=26), black line, or
E2J3 heterozygous (n=38), grey line, were aged and monitored for tumors.
800
Table 1.
Alive at
18 months
Age (days)
50% survival
Mean
age (days)
42.3%
521
530
53.8%
30.8%
557
557
509
503
57.9%
582
563
81.3%
40.9%
646
619
512
-
E2f3 wildtype (n=26)
Male (n=13)
Female (n=13)
E2f3 heterozygous (n=38)
Male (n=16)
Female (n=22)
517
Table 1. Survival data for p53 heterozygous mice depends on both E2f3 status and
gender. The percentage of mice surviving at 18 months, the age (days) at which half
the cohort have died, and the mean lifespan (days) of mice are determined.
alive, which shows a difference of two months. However, a more complicated picture
emerges when gender is also factored in (Figure 2). In this study, male p53 heterozygous
mice appear to survive longer than female p53 heterozygous mice. E2f3 status has little
effect on the survival of female p53 heterozygous mice. In contrast, the survival of male
E2f3,p53 double heterozygous mice is significantly enhanced relative to other mice in
this study. The phenotype of the male E2f3,p53 double heterozygous mice appears to be
specifically responsible for the difference in survival that is observed due to E2f3 status
when gender is not taken into account.
The tumor spectrum of p53 heterozygous mice in this study included soft tissue
sarcomas, osteosarcomas, carcinomas, and haematological tumors (Table 2,3). E2f3
heterozygosity altered the frequency of specific tumor sub-types. Fewer soft tissue
sarcomas were observed in E2f3;p53 double heterozygous mice, while there was an
increased incidence of haematological tumors in E2f3;p53 double heterozygous mice.
Closer examination of individual haematological tumor types revealed a significant
increase in histiocytic sarcoma and a reduction in generalized lymphomas.
E2f3 heterozygosity has been previously shown to affect tumor development
(Ziebold et al., 2003). However, it is unclear by what mechanism this is accomplished.
To date, all known cellular functions of E2F3 have been characterized comparing wild-
type and E2f3 knockout cells. E2F3 may possess functions relevant to tumorigenesis for
which it is haplo-insufficient.
It is also a possibility that E2f3 dosage is critical in certain
types of tumors, and that E2f3 heterozygosity results in a partial phenotype. Analysis of
the p53 heterozygous mice that are E2f3 knockout will help determine this possibility.
197
Figure 2.
1
0.9
Od3
.>
0.8
0.7
0.6
0,.5
cD
Ct
0.4
Q) 0.3
,.
0.2
C(1
0.1
0
0
100
200
300
400
500
600
700
days
- -*- E2f3 wildtype male (n = 13)
-- E2f3 wildtype female (n = 13)
- -*- - E2f3 heterozygous male (n = 16)
- E2f3 heterozygous female (n= 22)
Figure 2. E2f3 heterozygosity extends the lifespan of male p53 heterozygous mice.
Survival of p53 heterozygous mice is shown by E2f3 status and gender. Male
wildtype E2f3 (n=13), black dashed line; female wildtype E2f3 (n=13), black solid
line; male E2f3 heterozygous (n=16), grey dashed line; female E2f3 heterozygous
(n=22), grey solid line.
800
Table 2.
E2f3 wildtype
Gender
Age
Tumor
F:
M
M
F:
M
N
F
F
Fl
F
F
F'
F
M
F
M
F
F
F
vI
F
M
M
M
M
M
251
334
402
414
415
454
465
465
479
497
501
517
521
528
547
557
565
588
594
598
612
653
684
691
712
740
histiocytic sarcoma
UNDIAGNOSED
rhabdomyosarcoma
thymic lymphoma
NO HISTOLOGY
UNDIAGNOSED
unknown
UNDIAGNOSED
ductal pancreaticcarcinoma,pulmonary carcinoma
unknown
lymphoma (general)
leiomyosarcoma
thymic lymphoma
lymphoma (general)
osteosarcoma
UNDIAGNOSED
unknown
histiocytic sarcoma, mammary carcinoma
UNDIAGNOSED
NOHISTOLOGY
lymphoma (general), liposarcoma,leiomyosarcoma
hemangiosarcoma
pulmonary adenocarcinoma,hepatocellualrcarcinoma, leiomyosarcoma
lymphoma (general)
hemangiosarcoma
lymphoma (general)
E2f3 heterozygous
Gender
Age
Tumor
F
F
M
F
F
F
F
F
F
F
F
M
F
F
M
F
F
F
F
M
F
F
F
M
F
M
M
M
M
M
M
F
M
M
M
F
M
M
221
243
340
426
427
452
458
473
474
477
482
487
509
509
546
547
570
572
582
588
596
601
601
605
611
619
627
642
646
670
680
687
697
706
725
746
756
806
thymic lymphoma
NOHISTOLOGY
histiocytic sarcoma
osteosarcoma
NOHISTOLOGY
UNDIAGNOSED
NOHISTOLOGY
unknown
unknown
lymphoma (general)
pulmonary carcinoma
liposarcoma
UNDIAGNOSED
UNDIAGNOSED
pulmonary carcinoma
lymphoma (general)
thymic lymphoma
pulmonarycarcinoma
UNDIAGNOSED
NOHISTOLOGY
osteosarcoma
histiocytic sarcoma
histiocytic sarcoma
histiocytic sarcoma
histiocytic sarcoma
histiocytic sarcoma
histiocytic sarcoma
histiocytic sarcoma
rhabdomyosarcoma
hepatocellularcarcinoma, squamouscell carcinoma
hemangiosarcoma
UNDIAGNOSED
UNDIAGNOSED
histiocytic sarcoma
hepatocellularcarcinoma,osteosarcoma
UNDIAGNOSED
STILLALIVE
histiocytic sarcoma
Table 2. E2f3 status, gender, age (days) at death and pathology of p53 heterozygous
mice.,
Table 3.
E2f3
E2J3
Trumortype:
wildtype (n=26)
heterozygous (n=38)
SOFT TISSUE SARCOMA
7 (27%)
2 (8%)
3 (8%)
--hemangiosarcoma
--leiomyosarcoma
--liposarcoma
--rhabdomyosarcoma
1(3%)
3 (12%)
0 (0%)
1 (4%)
1(3%)
1 (4%)
1 (3%)
OSTEOSARCOMA
1 (4%)
CARCINOMA
-hepatocelluar carcinoma
-pulmonary carcinoma
-mammary carcinaoma
-ductal pancreatic carcinoma
-squamous cell carcinoma
5 (19%)
HAEMATOLOGICAL
-histiocytic sarcoma
-lymphoma (general)
-thymic lymphoma
7 (27%)
2 (8%)
5 (19%)
2 (7%)
14 (37%)
UNKNOWN
8
8
NO HISTOLOGY
2
4
STILL ALIVE
0
1
3 (8%)
6(16%)
1 (4%)
2 (5%)
2 (8%)
1 (4%)
3 (8%)
0 (0%)
0 (0%)
0 (0%)
1
1 (4%)
(3%)
10 (26%)
2
2
(5%)
(5%)
Table 3. E2f3 status alters the tumor spectrum of p53 heterozygous mice.
The gender bias observed among p53 heterozygous mice has not been previously
described (Attardi and Jacks, 1999). In fact, few tumor models have reported a gender
bias, despite the fact that gender differences are found in many human cancers(Jemal et
al., 2004). It is unclear what contribution strain background has in this study, which
could explain why these differences have not been noticed by others. Additionally, the
contribution of E2f3 status to this gender bias is interesting. In other mouse tumor
models, E2f3 has not been reported to affect tumorigenesis in a gender specific manner.
However, many of the phenotypes associated with E2f3-loss suggest that hormone and
growth factor signaling may be a contributing factor.
201
References.
Attardi, L. D., and Jacks, T. (1999). The role of p53 in tumour suppression: lessons from
mouse models. Cell Mol Life Sci 55, 48-63.
Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr.,
Butel, J. S., and Bradley, A. (1992). Mice deficient for p53 are developmentally normal
but susceptible to spontaneous tumours. Nature 356, 215-221.
Harvey, M., McArthur, M. J., Montgomery, C. A., Jr., Butel, J. S., Bradley, A., and
Donehower, L. A. (1993). Spontaneous and carcinogen-induced tumorigenesis in p53-
deficient mice. Nat Genet 5, 225-229.
Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, R. T.,
and Weinberg, R. A. (1994). Tumor spectrum analysis in p53-mutant mice. Curr Biol 4,
1-7.
Jemal, A., Tiwari, R. C., Murray, T., Ghafoor, A., Samuels, A., Ward, E., Feuer, E. J.,
and Thun, M. J. (2004). Cancer statistics, 2004. CA Cancer J Clin 54, 8-29.
Lang, G. A., Iwakuma, T., Suh, Y. A., Liu, G., Rao, V. A., Parant, J. M., Valentin-Vega,
Y. A., Terzian, T., Caldwell, L. C., Strong, L. C., et al. (2004). Gain of function of a p53
hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119, 861-872.
Olive, K. P., Tuveson, D. A., Ruhe, Z. C., Yin, B., Willis, N. A., Bronson, R. T.,
Crowley, D., and Jacks, T. (2004). Mutant p53 gain of function in two mouse models of
Li-Fraumeni syndrome. Cell 119, 847-860.
Purdie, C. A., Harrison, D. J., Peter, A., Dobbie, L., White, S., Howie, S. E., Salter, D.
M., Bird, C. C., Wyllie, A. H., Hooper, M. L., and et al. (1994). Tumour incidence,
spectrum and ploidy in mice with a large deletion in the p53 gene. Oncogene 9, 603-609.
Ziebold, U., Lee, E. Y., Bronson, R. T., and Lees, J. A. (2003). E2F3 loss has opposing
effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but
metastasis of medullary thyroid carcinomas. Mol Cell Biol 23, 6542-6552.
202
Appendix D
E2J3-loss impairs cell cycle exit
Aaron Aslanian and Jacqueline A. Lees
Author's contributions: Figure 1 and 2; Table 1 and 2
203
The Rb pathway has a key role in the regulation of cellular proliferation.
It has an
equally important role in cell cycle exit (Dannenberg et al., 2000; Sage et al., 2000).
Withdrawal from the cell cycle requires the ability to recognize various exit cues and
respond by increasing the expression of anti-proliferative genes and decreasing the
expression of pro-proliferative genes.
Cell cycle exit can either be a temporary state or a permanent state. At the
restriction point in late G1, cells monitor a variety of external cues such as growth factor
levels, extracellular matrix attachment and cell-cell contract. If conditions are
unfavorable, cells withdraw from the cell cycle and enter a GO/Gi state known as
quiescence. MEFs lacking all three pocket proteins do not have this response and instead
continue to proliferate. Primary cells undergo a finite number of cell divisions before
they permanently withdraw from the cell cycle and enter a state termed senescence.
MEFs lacking all three pocket proteins are immortal, they do not undergo senescence.
Differentiation also involves permanent cell cycle exit, as well as the initiation of a cell
type specific pattern of gene expression. Pocket proteins play a role in cell cycle exit
during differentiation and in some cases they are also involved in regulating the
subsequent cell type specific gene expression.
Pocket proteins regulate cell cycle exit through E2F. This requires the inhibition
of the activating E2Fs as well as active repression. The latter activity is the role of the
repressive E2Fs, E2F4 and E2F5. These E2Fs have been shown to associate with the
promoters of genes along with pocket proteins and chromatin modifying enzymes
(Macaluso et al., 2003; Rayman et al., 2002). MEFs lacking E2f4 and E2f5 fail to
respond to several cell cycle exit cues, including expression of p16INK4A (Gaubatz et
204
al., 2000). They are also required as cofactors of the Smads for the repression of c-myc in
response to TGF-3 expression (Chen et al., 2002). Finally, expression of a carboxyterminal E2F mutant lacking the pocket protein binding domain and the transactivation
domain led to significant derepression of many E2F target genes, although this study did
not identify the E2Fs normally responsible for regulation of these genes (Rowland et al.,
2002).
Recently, the E2f3 locus was discovered to specify a second protein, E2F3B,
which associates with pRb in quiescent cells (Leone et al., 2000). E2F3B expression
does not vary during the cell cycle, mirroring that of the repressive E2Fs. We have
demonstrated that E2F3B is involved in the repression of at least one E2F target gene,
p19ARF (Aslanian et al., 2004). Given the levels of E2F3B present in quiescent cells,
and its association with pRb, we reasoned that E2F3B might play a role in cell cycle exit
and the repression of E2F target genes.
To ascertain whether E2F3 was involved in cell cycle exit, we monitored cell
cycle arrest initiated by contact inhibition. Although the onset of confluence was delayed
in E2f3 knockout MEFs because of their proliferation impairment, they eventually
appeared to exit the cell cycle (Figure 1A). However, they often achieved a higher cell
density than wild-type cells at confluence (Figure 1A,B). This was particularly surprising
given the increased cell size of E2f3 knockout MEFs (Appendix A), implying that cell
number should be decreased at confluence. Examination of confluent E2f3 knockout
MEFs revealed multiple layers of cells (data not shown). However, E2f3 knockout MEFs
did not show any other characteristics associated with transformation. Preliminary
examination of gene expression revealed that confluent wild-type MEFs decrease
205
Figure 1.
A)
60000000
- 50000000
T dd
t 40000000
h
: 30000000
-- 20000000
) 10000000
0
0
2
4
6
8
10
days
*
B)
wildtype
-
- E2f3 knockout
wildtype
E2f3 knockout
Figure 1. E2f3 knockout MEFs reach a higher saturation density than wild-type MEFs.
(A) Proliferation of wildtype and E2f3 knockout MEFs was monitored for 10 days. (B)
Wildtype and E2f3 knockout MEFs were stained with Modified Wright Giemsa Stain
(Sigma WG32) after 10 days to visualize cell density.
expression of E2F3A but not E2F3B (data not shown). This suggests that it is the
absence of E2F3B in these cells that affects their ability to exit the cell cycle. It will be
interesting to examine whether E2f3b knockout MEFs show a stronger contact inhibition
defect.
We subsequently determined whether E2F3 was required for cell cycle arrest
following serum deprivation. E2f3 knockout MEFs continue to incorporate BrdU even
after three days in low serum containing media (data not shown). We reasoned that cells
that were unable to enter quiescence might be more responsive to media with less than
10% serum. Wild-type and E2Jf3 knockout MEFs that were grown in low serum for three
days were then stimulated with 0.1%, 1% or 10% serum containing media and cell cycle
re-entry was monitored (Figure 2A). Wild-type MEFs re-enter the cell cycle in 10%
media but do not at the lower serum concentrations.
In contrast, E2f3 knockout MEFs re-
enter the cell cycle to a similar degree when stimulated with either 1% or 10% serum
containing media. This suggests that quiescent E2f3-deficient MEFs are more permissive
to cell cycle re-entry.
These observations regarding low serum growth are intriguing given our previous
observations that the loss of E2f3 impairs cell cycle re-entry. It indicates that E2F3 has
two roles; E2F3 is required for inhibiting proliferation in quiescent cells and then
required again for promoting proliferation during cell cycle re-entry. Preliminary
examination of E2F target genes did not reveal a consistent pattern to explain this data
(Figure 2B). Determination of the role of E2F3 in quiescent cells can be accomplished
by the identification of E2F3 target genes using ChIP. Comparison of these genes with
those identified by the same method to be regulated by E2F4 could yield a set of E2F3-
207
Figure 2.
A)
200000
0
cO
o
o
. CL,
150000
100000
50000
0
0
4
8
12
16
20
24
hours
I
wildtype
- -- -E2f3 knockout
200000
0 o
1% serum
150000
i0;
100000
,
50000
E
.lU
II
I
I I
M *-
0
I-
0
-
4
I-
-
I.
I
-
8
~&V I
12
16
1:
I
I
I
20
24
hours
*
- 4- -E2f3 knockout
wildtype
200000
01
0.1% serum
· "C0
.
150000
-q
0d
f 100000
1=
O1
·QC50000
E:
0
I
0
4
8
12
16
20
24
hours
-- wildtype - 4- -E2f3knockout
Figure 2. continued
B)
10% serum
1% serum
wildtype
c
E2f3 knockout
O q N
c
,---,--- Cq1
c O ',
wildtype
E2f3 knockout
O C q
--.,--. C',c
ll] .
I
-
{3-tubulin
W*' ;
-_
*
A- of p
phospho-Akt
T
UI0u
_-
phospho-Erk
_--WI
.
"Ilw"Niow
- -
p107
cyclin A
w~_
-.
e4S 4m
-
-_
A&MI..
MW
adWiO*
cdc2
i
p21CIP
Figure 2. Quiescent E2f3 knockout MEFs are permissive to cell cycle re-entry. (A)
Wildtype (solid black lines) and E2f3 knockout MEFs (dotted black lines) were grown in
low serum for three days. They were then stimulated to re-enter the cell cycle with media
containing either 10 percent, 1 percent or 0.1 percent serum. Cell cycle re-entry was
monitored by 3H-thymidine incorporation. (B) Whole cell extracts were prepared from
the above experiment and western blotted for P-tubulin (Sigma T4026), phospho-Akt
(Cell Signaling 9271), phospho-Erk (Cell Signaling 9101), p107 (sc-318), cyclin A (sc596), cdc2 (sc-54), and p21CIP (sc-6246).
specific genes. E2f3a and E2f3b specific knockout MEFs will also be useful in this
endeavor.
Given the prominent role of E2F4 in E2F target gene repression, we reasoned that
E2f3 and E2J4 might cooperate to repress certain E2F target genes. It will be interesting
to examine whether E2F3B participates in cell cycle exit in response to p16INK4A and
TGF-(3 expression. To begin to address whether E2F3B and E2F4 have overlapping
roles, we crossed E2f3 and E2f4 knockout mice to generate compound mutant animals.
Both E2f3 and E2f4 knockout mice are viable in a mixed genetic background (Humbert et
al., 2000a; Humbert et al., 2000b). However, E2fl;E2f3 compound mutant animals do
not survive beyond e9.5, and the conclusion drawn from this result was that the combined
loss of activating E2Fs was responsible for this phenotype (Cloud et al., 2002).
E2f3;E2f4 compound mutant animals also display reduced viability, although the exact
window of lethality was not determined (Table 1,2). This phenotype can be attributed to
loss of repression, but this cross will need to be repeated using E2f3b specific knockout
mice. It will also be helpful to determine the individual patterns of expression of E2F3A
and E2F3B during development.
It has been suggested that pRb regulates a distinct subset of E2F target genes from
p107 and p130. Given the specificity of pocket protein E2F interaction, a similar division
of target genes may exist among E2Fs. Clearly, E2F3 plays a key role in both cell cycle
progression and cell cycle exit.
210
Table 1.
E2f4
+/+
+/-
-/
+/+
E2f3 +/-I-
Table 1. Progeny (n=92) from E2f3;E2f4 double heterozygous matings at e13.5.
Table 2.
E2f4
+/+ +/E2f3
-/
+/+
+/-I-
--
v
v
Table 2. Progeny (n=53) from E2f3;E2f4 double heterozygous matings at el 1.5.
References.
Aslanian, A., Iaquinta, P. J., Verona, R., and Lees, J. A. (2004). Repression of the Arf
tumor suppressor by E2F3 is required for normal cell cycle kinetics. Genes Dev 18, 14131422.
Chen, C. R., Kang, Y., Siegel, P. M., and Massague, J. (2002). E2F4/5 and p107 as Smad
cofactors linking the TGFbeta receptor to c-myc repression. Cell 110, 19-32.
Cloud, J. E., Rogers, C., Reza, T. L., Ziebold, U., Stone, J. R., Picard, M. H., Caron, A.
M., Bronson, R. T., and Lees, J. A. (2002). Mutant mouse models reveal the relative roles
of E2F1 and E2F3 in vivo. Mol Cell Biol 22, 2663-2672.
Dannenberg, J. H., van Rossum, A., Schuijff, L., and te Riele, H. (2000). Ablation of the
retinoblastoma gene family deregulates G(1) control causing immortalization and
increased cell turnover under growth-restricting conditions. Genes Dev 14, 3051-3064.
Gaubatz, S., Lindeman, G. J., Ishida, S., Jakoi, L., Nevins, J. R., Livingston, D. M., and
Rempel, R. E. (2000). E2F4 and E2F5 play an essential role in pocket protein-mediated
G1 control. Mol Cell 6, 729-735.
Humbert, P. O., Rogers, C., Ganiatsas, S., Landsberg, R. L., Trimarchi, J. M., Dandapani,
S., Brugnara, C., Erdman, S., Schrenzel, M., Bronson, R. T., and Lees, J. A. (2000a).
E2F4 is essential for normal erythrocyte maturation and neonatal viability. Mol Cell 6,
281-291.
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A.
(2000b). E2f3 is critical for normal cellular proliferation. Genes Dev 14, 690-703.
Leone, G., Nuckolls, F., Ishida, S., Adams, M., Sears, R., Jakoi, L., Miron, A., and
Nevins, J. R. (2000). Identification of a novel E2F3 product suggests a mechanism for
determining specificity of repression by Rb proteins. Mol Cell Biol 20, 3626-3632.
Macaluso, M., Cinti, C., Russo, G., Russo, A., and Giordano, A. (2003). pRb2/p130E2F4/5-HDAC 1-SUV39H 1-p300 and pRb2/p 130-E2F4/5-HDAC 1-SUV39H 1-DNMT 1
multimolecular complexes mediate the transcription of estrogen receptor-alpha in breast
cancer. Oncogene 22, 3511-3517.
Rayman, J. B., Takahashi, Y., Indjeian, V. B., Dannenberg, J. H., Catchpole, S., Watson,
R. J., te Riele, H., and Dynlacht, B. D. (2002). E2F mediates cell cycle-dependent
transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor
complex. Genes Dev 16, 933-947.
Rowland, B. D., Denissov, S. G., Douma, S., Stunnenberg, H. G., Bernards, R., and
Peeper, D. S. (2002). E2F transcriptional repressor complexes are critical downstream
targets of pl9(ARF)/p53-induced proliferative arrest. Cancer Cell 2, 55-65.
213
Sage, J., Mulligan, G. J., Attardi, L. D., Miller, A., Chen, S., Williams, B., Theodorou, E.,
and Jacks, T. (2000). Targeted disruption of the three Rb-related genes leads to loss of
G(1) control and immortalization. Genes Dev 14, 3037-3050.
214
Appendix E
The retinoblastoma protein:
Will the real tumor suppressor function please stand up?
Aaron Aslanian and Jacqueline A. Lees
215
PREFACE
The retinoblastoma protein (pRb) has been shown to provide key cellular
activities regulating the proliferation of cycling cells, the ability of cells to effectively exit
the cell cycle, and the execution of tissue-specific differentiation programs. Despite this
knowledge, we have yet to pinpoint the role(s) of pRb that make it a critical tumor
suppressor protein. This, in part, reflects our evolving understanding of how cancers
arise. The recent generation of conditional, tissue-specific deletions of the
retinoblastoma (Rb) gene are finally allowing us to probe the function of pRb in different
cell types and should be a critical step in uncovering the cellular genesis of cancers.
INTRODUCTION
Retinoblastoma is a hereditary childhood cancer in which affected individuals
possess one germline mutation of the retinoblastoma gene. The highly probable mutation
of the remaining copy of the gene usually leads to the development of bilateral cancer.
Sporadic cases are also observed in the form of unilateral retinoblastoma, resulting from
two somatic mutations in Rb. The fact that inactivation of Rb is both necessary and
sufficient for retinoblastoma formation has made it a useful model for understanding
tumor suppressor gene function.
Although current medical techniques make the detection and removal of
retinoblastoma a non-lifethreatening cancer, individuals with germline mutation are also
predisposed to several other types of tumors, especially osteosarcomas (Fletcher et al.,
2004). Additionally, inactivation of the retinoblastoma gene is known to occur is many
216
cancers, including small cell lung carcinoma, cervical carcinoma, prostate carcinoma,
breast carcinoma, and bladder carcinoma. Therefore, pRb clearly has functions that
broadly affect a number of different cell types.
In the almost 20 years since it was first cloned, pRb has been shown to interact
with over 100 different proteins and has been linked to over a score of different cellular
processes (Morris and Dyson, 2001). Despite this wealth of information on pRb function,
the explicit tumor suppressive role of pRb remains unclear. Complicating matters are
two related proteins, p107 and p130 (Classon and Dyson, 2001; Claudio et al., 2002;
Paggi and Giordano, 2001). While p107 and 130 are themselves infrequently targeted in
cancers, mutation of upstream regulators of pRb as well as transforming viral
oncoproteins nevertheless functionally inactivate all three proteins. Additionally, the
preferred mode of pRb inactivation varies among different types of cancer, suggesting
that the critical function of pRb may be highly dependent on cellular context. Because
p107 and p130 can sometimes share pRb function, it will be important to understand the
unique properties of pRb that specifically make it a tumor suppressor protein and under
what circumstances p107 and p130 can play a "back-up" tumor suppressor role.
THE POCKET PROTEIN FAMILY
pRb, p107 and p130 comprise the pocket protein family, so named for a
conserved region through which many of their known interactions and functions occur.
The small pocket consists of an A domain and a B domain separated by a spacer region.
It binds to an LxCxE motif found in many known pocket protein interactors. Viral
oncoproteins that disrupt pocket protein function also bind the small pocket through an
217
I,xCxE motif (see Text Box 1). Whether LxCxE-containing proteins associate with all
three pocket proteins to a similar degree has not been addressed in most cases.
Additionally, the spacer region of p10O7and p130 contains a motif that bares similarity to
cyclin dependent kinase (cdk) inhibitors (Castano et al., 1998; Woo et al., 1997; Zhu et
al., 1995). Through this motif, p107 and p130 can associate with and inhibit cyclin-cdk
complexes.
Indeed, p1O7 has been shown to play a role in down-regulating cdk activity,
although the biological circumstances under which this function may be relevant remain
unclear (Chibazakura et al., 2004). p107 and p130 are more closely related to each other
than either is to pRb, suggesting that structural differences outside the small pocket may
influence the tumor suppressive function among the pocket proteins.
The expression pattern of the three pocket proteins is also differentially regulated.
The expression of pRb does not vary significantly during the cell cycle or upon cell cycle
exit. On the other hand, p10O7,which is an E2F target gene, is not expressed in quiescent
cells and becomes induced as cells proceed through S-phase. In contrast, p130 is highly
expressed in quiescent and differentiated cells. Upon cell cycle re-entry, p130 levels
decrease as a result of ubiquitin-mediated degradation by the proteasome (Bhattacharya
et al., 2003; Tedesco et al., 2002). Regulation of pRb appears to occur primarily via
phosphorylation (see Text Box 2). In GO and Gi cells, pRb is present in a hypo-
phosphorylated state. Sequential phosphorylation events by cyclin-cdk complexes during
cell cycle progression lead to the accumulation of hyper-phosphorylated
pRb. This
change in phosphorylation status affects the ability of pRb to associate with many of its
protein interactors. Mutation of cdk phosphorylation sites on pRb results in a
constitutively active pRb (Knudsen and Wang, 1997). Both p107 and p130 are also
218
substrates for cyclin-cdk phosphorylation, although the relative importance of
phosphorylation in relation to other modes of regulation for these two proteins is unclear.
Pocket proteins can both positively and negatively regulate the activities of
proteins with which they associate, in order to control gene expression that affects cell
cycle progression, cell cycle exit and differentiation.
To this end, pocket proteins also
interact with different chromatin and histone modifying enzymes to actively regulate
gene expression, while in other cases sequestration is sufficient to passively regulate gene
expression. For example, chromatin immunopercipitation (ChIP) experiments suggest
that p107 and p130, but not pRb, directly bind the promoters of cell cycle genes. Instead,
pRb can be found only at the promoters of target genes involved in differentiation.
Additionally, differences in the stability of different pocket protein complexes indicate a
distinct role for pRb compared to p107 and p130 in the regulation of gene expression
(Sullivan et al., 2004; Young and Longmore, 2004). These studies suggest that pRb may
bind to target gene promoters to stably impact gene expression, while p107 and p130
transiently regulate target gene promoters. Discussed below are several key pRb
interacting proteins and their role in the regulation of gene expression.
CELL CYCLE PROGRESSION
The most well studied role of pRb in the regulation of cell cycle progression
occurs through its interaction with the E2F family of transcription factors (Trimarchi and
Lees, 2002). The E2F transcription factor is a heterodimer, consisting of an E2F subunit
and a DP subunit. Functional specificity is provided by the E2F subunit. Eight E2Fs
have been identified to date, although only five interact with the pocket proteins.
219
Endogenous E2Fs and pocket proteins exhibit specificity among their preferred proteinprotein interactions. pRb interacts with E2F1, E2F2, and E2F3; p107 interacts primarily
with E2F4; and p130 interacts with both E2F4 and E2F5. pRb interacts with E2F
transcription factors in its hypo-phosphorylated
state and in its hyper-phosphorylated
state pRb releases free E2F.
The E2F transcription factors are a family of non-LxCxE containing proteins that
interact with the large pocket of pRb, consisting of the small pocket and an additional C
domain. E2F1, E2F2, and E2F3A are strong transcriptional activators regulated
specifically by pRb. It has been shown that pRb can induce passive repression via
sequestration and inhibition of E2F transactivation capacity (Helin et al., 1993). E2F4
and E2F5 are poor transcriptional activators, and they are found primarily in the
cytoplasm when not bound by pocket proteins. For these reasons, E2F4 and E2F5 are
believed to mainly function in pocket protein mediated repression (Bruce et al., 2000;
Chen et al., 2002; Gaubatz et al., 2000; Rayman et al., 2002). E2F3B is also thought to
function as a transcriptional repressor, although this has not been extensively
characterized (Aslanian et al., 2004; Leone et al., 2000).
Classic E2F target genes regulate processes such as cell cycle progression and
DNA replication. Pocket proteins cooperate with E2Fs to repress their expression during
GO and G1, and the release of free E2F permits the induction of E2F target gene
expression.
While early experiments focused on the role of E2F target genes in the G1/S
transition, novel techniques involving microarray and chomatin immunopercipitation
(ChIP) have greatly expanded the list of putative E2F target genes, as well as expand
their functional roles. Mad2 is an example of one such gene involved in mitotic
220
progression, whose expression is deregulated in Rb-deficient cells (Hernando et al.,
2004). Another study identified Skp2 deregulation in plO07-deficientcells, although this
may not be direct (Rodier et al., 2005). It has been suggested that pRb and its pocket
protein family members p107 and p130 normally regulate distinct subsets of E2F target
genes, which may contribute to their differential status in cancer (Classon et al., 2000).
Mouse models have been helpful to illustrate the importance of E2F as a critical
player in pRb-mediated cell cycle regulation. Rb-deficient embryos display inappropriate
proliferation in several tissues and this defect appears to be cell autonomous (de Bruin et
al., 2003; Jacks et al., 1992; Lee et al., 1992; Wu et al., 2003). Simultaneous deletion of
Rb and either E2fl or E2f3 suppresses this phenotype, suggesting that the release of
activating E2Fs is a key consequence of Rb-loss (Tsai et al., 1998; Ziebold et al., 2001).
This is also true in tumorigenesis.
Unlike their human counterparts, mice heterozygous
for RB succumb to pituitary and thyroid tumors (Jacks et al., 1992). The additional
deletion of either E2fl or E2f3 delays the onset of tumorigenesis, resulting in a significant
extension of lifespan (Yamasaki et al., 1998; Ziebold et al., 2003).
Surprisingly, the loss of E2f4 also results in an extension of lifespan of Rb
heterozygous mice (Lee et al., 2002). Careful analysis of both cells and tumors deficient
for E2f4 and Rb demonstrated that the composition of the remaining E2F and pocket
protein complexes underwent reshuffling. In this setting, p107 and p130 bind to E2F3
and E2F1 respectively, correcting the release of free activating E2Fs. This demonstrates
thatplO7 and p130 are capable of performing a "back-up" tumor suppressor role in the
absence of pRb. It is interesting to note that the loss of E2f4 does not rescue
221
inappropriate proliferation in Rb-null embryos, suggesting that reshuffling may not occur
in this setting.
Despite the attention given to E2F, other RB proteins interactions also affect cell
cycle progression. The Id family is one such example. Id2 has been shown to bind to
pRb, as well as p107 and p130 (Lasorella et al., 1996). In quiescent cells, d2 is in a
complex with p130 that persists during G1. During S-phase, Id2 associates with both
pRb and p107. The loss of Id2, which is highly expressed in neuronal and
haematopoietic cells, extends the lifespan of Rb-deficient embryos (Iavarone et al., 2004).
Additionally, the loss of Id2 leads to increased survival of Rb heterozygous mice
(Lasorella et al., 2005). This demonstrates that the deregulation of multiple pRb
interacting proteins can contribute to altered gene expression and tumorigenesis.
pRb not only regulates RNA polymerase II-dependent transcription, but also
affects the activity of RNA polymerase I and III (White, 2004; White, 2005). Both pRb
and p130, but not p107, interact with UBF to repress rDNA transcription (Hannan et al.,
2000a; Hannan et al., 2000b; Voit et al., 1997). Cells deficient for both these pocket
proteins have abnormally elevated rDNA levels and conversely over-expression of either
pRb or p130 represses rDNA transcription.
UBF binds to the small pocket of pRb and
p130, and given the significant contribution of rDNA transcription to the total
transcription, may represent an important growth control mechanism.
Similarly, pRb is
capable of interacting with TFIIIB, part of the general transcriptional machinery of
polymerase III-dependent promoters (Chu et al., 1997; Hirsch et al., 2000). This
interaction maps to the large pocket. pRb also has been shown to repress the U6
promoter and tRNA levels are elevated in many tumors (Hirsch et al., 2004). Because
222
tRNA can be potentially rate limiting for protein synthesis, this may represent an
important target in tumorigenesis.
In addition to regulating the activity of various transcription factors, pRb also
plays more direct role at origins during DNA replication. This work comes in part from
studies involving Drosophila melanogaster, where some origins have been defined. Rbf,
the Drosophila pRb homolog, can be found in complex with DmORC (Bosco et al.,
2001). Interestingly, this complex also includes dE2F1 and dDP, suggesting that E2F-
binding activity may be required for this localization. It is unclear whether other
transcription factors interacting with pRb bring it to additional origins of replication.
DNA replication factors interact with pRb, which may suggest a reason for pRb
localization to DNA during replication (Schmitz et al., 2004). Additionally, pRb
regulates histone gene synthesis required for chromatin assembly (Gupta et al., 2003;
Herrera et al., 1996; Lemercier et al., 2000). Finally, pRb has also been localized to sites
of DNA damage, and may bring interacting proteins to DNA in this context as well (Avni
et al., 2003).
CELL CYCLE EXIT
In addition to playing a key role in events during the cell cycle, the pocket
proteins also have an important function in the decision to withdrawal from the cell cycle.
Cells integrate a number of cues, both extracellular and intracellular, when making the
decision to exit the cell cycle. In mammalian cells, the restriction point in late G1 is
defined as the point after which cells are committed to replicate their DNA and divide.
Once past the restriction point, cell cycle progression is no longer dependent on external
223
cues such as serum, matrix attachment, and cell-cell contact. The pocket proteins appear
to lie at the crux of many of these decisions. Importantly, most transformed cells are no
longer responsive to these cues.
Quiescence is a temporary GO/G1 state that cells enter if conditions do allow them
to proceed through the restriction point. Once growth conditions again become
favorable, cells re-enter the cell cycle. Acute ablation of Rb but not germline deletion
renders cells incompetent to exit the cell cycle in response to serum deprivation (Sage et
al., 2003). This may be explained by upregulation of p107 observed in the germline
deletion of Rb, and the ability of p107 to compensate for pRb in this setting. Consistent
with this theory, germline deletion of both Rb and p107 results in bypass of the serumdependent restriction point while maintaining the anchorage-dependent checkpoint (Gad
et al., 2004). Cells deficient for all three pocket proteins fail to arrest in response to
serum deprivation, contact inhibition or the loss of adhesion (Dannenberg et al., 2000;
Sage et al., 2000).
Mammalian cells undergo a more permanent arrest termed senescence following a
defined number of population doublings due to their finite replicative potential. Many
factors including telomere shortening and DNA damage caused by reactive oxygen
species can influence the timing of senescence. However, all of these signals appear to
ultimately funnel through the pocket proteins. Inactivation of all three pocket proteins,
either through genetic knockout or viral oncogenes, renders cells immortal (Dannenberg
et al., 2000; Sage et al., 2000). Reintroduction of Rb into Rb-deficient cell lines induces
a senescence-like state. Additionally, the acute loss of Rb in senescent cells is sufficient
allow them to re-enter the cell cycle (Sage et al., 2003). From a broader perspective,
224
senescence may be part of a tumor surveillance mechanism and so it is no surprise that
pRb plays a central role in it.
Differentiation not only involves permanent withdrawal from the cell cycle, but
also the execution of a tissue-specific pattern of gene expression.
Many recent studies
have implicated the pocket proteins in an active role in promoting differentiation-specific
gene expression. However, the dual roles of controlling cell cycle exit and differentiation
have clouded the specific roles of the pocket proteins in these processes.
DIFFERENTIATION
Mouse models have implicated pRb and the other pocket proteins in the process
of differentiation.
One of the long-standing interests in the cancer field has been to
understand the role of pRb in the retina, and how its absence leads to retinoblastoma.
The fact that Rb heterozygous mice do not mirror their human counterparts was
disappointing (Jacks et al., 1992). Rb-null chimeric mice also succumb to pituitary and
thyroid tumors, but show no evidence of retinoblastoma (Williams et al., 1994).
Recently, tissue specific deletion of Rb has allowed further exploration of its role in
retinal differentiation and development.
Conditional knockout of Rb in the retina of p0l7-deficient mice resulted in
enhanced proliferation of retinal precursor cells (Chen et al., 2004; MacPherson et al.,
2004). The increase in inappropriate proliferation is accompanied by elevated apoptosis,
but most cells eventually exited the cell cycle. Similar results were obtained through the
conditional knockout of Rb in p130-deficient mice, demonstrating that either of the two
remaining pocket protein family members can potentially compensate for the loss of pRb.
225
The next step in these studies will be in understanding how pRb and the other pocket
proteins specify retinal development, and in particular how this proceeds among the
many different cellular subtypes found within the retina.
Germline deletion of Rb causes embryonic defects in both the central nervous
system (CNS) and the peripheral nervous system (PNS), which stimulated interest in the
role of pRb in neuronal cells types (Jacks et al., 1992; Lee et al., 1992). Subsequent
generation of chimeric mice as well as conditional deletion of Rb helped to demonstrate
that inappropriate S-phase entry and elevated E2F activity observed in the CNS are cell
autonomous defects (Lipinski et al., 2001). One striking phenotype due to conditional
deletion of Rb in the CNS is increased brain size due to increased neurogenesis (Ferguson
et al., 2002; MacPherson et al., 2003). Consistent with the above observations, Rb-null
neuronal stem cells display a delay in cell cycle exit when stimulated to differentiate
(Callaghan et al., 1999). Additionally, p107 was found complexed with E2F3, suggesting
that p107 can partially compensate for Rb-loss in this setting.
Conditional deletion of Rb has also been used to probe the role of pRb in
development and tumorigenesis of specific neuronal subtypes. Granule cells lacking Rb
display defects in cell cycle exit and differentiation (Marino et al., 2003). In contrast,
astrocytes and Purkinje cells lacking both Rb and p107 appear to undergo normal
terminal differentiation.
Inactivation of all three pocket proteins via viral oncoprotein
expression causes apoptosis in certain neuronal cell types. Mice in which Rb is
conditionally deleted using a GFAP-Cre develop medulloblastoma in a p53-null
background (Marino et al., 2000). GFAP-T121 expressing mice develop astrocytoma
(Xiao et al., 2002). Astrocytoma is not observed when only Rb is inactivated,
226
demonstrating that multiple pocket proteins function in this cell type. This is supported
by data on genetic mutations observed in astrocytoma.
Osteosarcomas are a common secondary tumor type that develops in Rb
heterozygous individuals (Fletcher et al., 2004). Although no mouse models exist
examining the role of Rb in osteosarcoma, pocket proteins have been connected to bone
development. Osteoblast differentiation requires the function of pRb (Thomas et al.,
2001; Thomas et al., 2004). Rb-deficient MEFs are unable to express late stage
osteogenic differentiation markers, while MEFs deficient for both p107 and p130
differentiate into osteoblasts normally. CBFA1/RUNX2 and pRb directly interact and
pRb can stimulate the transcriptional activity of CBFA1/RUNX2. This demonstrates that
pRb has a positive role in osteoblast differentiation.
Although p107 and p130 have not
been shown to play a role in osteoblast differentiation, they are critical in chondrocytes
(Cobrinik et al., 1996; Laplantine et al., 2002). Embryos deficient for p107 and p]30
display defects in chondrocyte development.
One of the earliest differentiation processes to be directly linked with pRb is
muscle. pRb has been shown to directly interact with the muscle-specific differentiation
factor MyoD(Novitch et al., 1996). MyoD is a member of the E-box transcription factor
family that is capable of inducing differentiation into muscle. pRb can enhance the
activity of myoD. Rb-deficient cells stably overexpressing myoD fail to upregulate late
stage markers of muscle differentiation and also fail to permanently exit the cell cycle
(Novitch et al., 1999). Recently, a role for pocket proteins in cardiac development has
also been uncovered (Jung et al., 2005; MacLellan et al., 2005; Papadimou et al., 2005).
227
Mouse models also support a cell autonomous role for pRb in muscle.
Conditional deletion of Rb results in embryonic survival until birth, but death shortly
thereafter due to an inability of diaphragm muscles to inflate the lungs. Closer
examination of skeletal muscle reveals multiple defects in musculature. Differentiation
of Rb-deficient myoblasts results in similar problems (Camarda et al., 2004; Huh et al.,
2004). However, deletion of Rb in differentiated myotubes appears to have no visible
consequences, suggesting the pRb is not essential for maintenance of differentiation.
Finally, deletion of N-ras in Rb-deficient embryos appears to rescue defects associated
with muscle differentiation/development,
although this is likely to be indirect (Takahashi
et al., 2003).
Besides the lens, neuronal tissue, bone and muscle, Rb has been shown to have
function in a wide range of other tissues also including adipocytes, lung, and the
haematopoetic compartment. In these different settings, pRb interacts with transcription
factors that specify different tissue-specific patterns of gene expression. It is not clear
whether they all involve a positive role for pRb in promoting differentiation or whether
the role of pRb is limited to cell cycle exit in some cases. As we gain greater
understanding;of Rb function in different tissues, this will improve our understanding of
the role of pRb as a tumor suppressor.
CONCLUSIONS
In our efforts to rein in cancer, it will be important to understand not only the
molecular changes that correlate with cancer, but also from where tumor cells arise.
Many of the signaling pathways co-opted by tumor cells are normally used during early
228
development to rapidly increase cell number and move cells to their appropriate residence
within the body. Several models have been offered to explain this connection.
Dedifferentiation is a popular model that proposes cancers arise from a once committed
cell that re-enters the cell cycle, perhaps losing some or all of its tissue specific gene
expression profile, and resumes growth. A model for tumorigenesis that has more
recently gained favor suggests that cancer may arise from populations that either are or
are similar to stem cells rather than more fully committed cells.
Great effort has been made to identify genes that specify stem cell character in
different tissues. There have also been some studies that have specifically examined the
Rb pathway in these cells. Embryonic stem (ES) cells cycle rapidly due to constitutive
cdk activity and hyper-phosphorylation
of pRb (Miura et al., 2004; Savatier et al., 1994;
Stead et al., 2002; White et al., 2005). This would indicate that pRb is inactive in these
cells, and only upon commitment to specific lineages is pRb function relevant. Some
recent studies confirm that pocket proteins have important functions in stem cell
populations (Callaghan et al., 1999; Jori et al., 2004; Papadimou et al., 2005). What role
pRb is playing in these lineages remains to be determined. The expansion of tissue
specific mouse models should accelerate our ability to answer these questions.
229
TEXTBOX
1:
Adenovirus, SV40 polyomavirus, and human papillomavirus (HPV) are small
DNA tumor viruses. Because of their small genome size, these viruses have opted to
utilize host cell machinery to carry out viral DNA replication. To this accomplish end,
the viruses must force the host cell to enter S-phase from a quiescent or otherwise postmitotic state. These features have made DNA tumor viruses a useful tool for early
studies investigating cellular DNA replication, cell cycle control, and cellular
transformation.
HPV has been associated with cervical cancer, but adenovirus and polyomavirus
have not been linked to any human cancers. However, all three viruses are capable of
causing the transformation of a wide variety of cell types. The adenoviral oncoprotein
E1A, HPV E7, and SV40 large T antigen each bind the pocket proteins via an LxCxE
motif, presumably disrupting many normal pocket protein interactions. This interaction
is essential for transformation by these viruses. Studies with viral oncoproteins were
responsible for providing key insights into pRb, leading to: (1) its identification as a
member of a family of proteins, (2) its role a regulator of the cell cycle, and (3) the
identification of a major target, the E2F transcription factor.
Interestingly, two additional families of tumor viruses, poxviruses and EpsteinBarr viruses (herpesvirus family) are known for which there is no direct evidence to
suggest that these viruses interfere with pRb function. Rather their genomes include
homologs of known E2F target genes such as ribonucleotide reductase, suggesting that
deregulation of proliferation is a key target for tumor viruses in terms of both viral
replication and the induction of tumorigenesis.
230
TEXTBOX 2:
Proper cell cycle progression depends on many carefully coordinated events to
orchestrate DNA replication and subsequent cell division. Cyclin-cyclin dependent
kinase (cdk) complexes set-up a number of key events in the cell cycle, and this is
conserved from yeast to human. In mammalian cells, pRb and its pocket protein family
members p107 and p130 are all substrates for cyclin-cdks. Pocket protein
phosphorylation leads to dissociation of E2F-pocket protein complexes and increased
E2F activity.
Cyclin D/cdk4,6 activity peaks during late Gi, cyclin E/cdk2 activity during G1/S
and then cyclin A/cdk2 activity peaks during late S-phase. Cyclin D is induced by
numerous growth factors, whereas cyclin E and cyclin A are both E2F target genes.
Some work has been done to examine specificity among different cyclin-cdk complexes
for various sites on the pocket proteins. Many experiments have made use a mutant pRb
protein in which nine phosphorylation sites have been mutated to alanines to render the
protein constitutively active.
Additional regulation of kinase activity occurs through the action of small
inhibitory proteins. The INK4 family (p16INK4A, pSINK4B, p18INK4C, p19INK4D)
and the CIP/KIP family (p21CIP, p27KIP, p57KIP2) inhibit cdk4,6 and cdk2
respectively.
CDK inhibitory proteins play key roles in initiating terminal cell cycle exit.
Also, p21CIP is a key p53 target gene involved in cell cycle arrest following genotoxic
stress, and provides an important link between these two tumor suppressor pathways.
231
References.
Aslanian, A., Iaquinta, P. J., Verona, R., and Lees, J. A. (2004). Repression of the Arf
tumor suppressor by E2F3 is required for normal cell cycle kinetics. Genes Dev 18, 14131422.
Avni, D., Yang, H., Martelli, F., Hofmann, F., ElShamy, W. M., Ganesan, S., Scully, R.,
and Livingston, D. M. (2003). Active localization of the retinoblastoma protein in
chromatin and its response to S phase DNA damage. Mol Cell 12, 735-746.
Bhattacharya, S., Garriga, J., Calbo, J., Yong, T., Haines, D. S., and Grana, X. (2003).
SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human
cells. Oncogene 22, 2443-2451.
Bosco, G., Du, W., and Orr-Weaver, T. L. (2001). DNA replication control through
interaction of E2F-RB and the origin recognition complex. Nat Cell Biol 3, 289-295.
Bruce, J. L., Hurford, R. K., Jr., Classon, M., Koh, J., and Dyson, N. (2000).
Requirements for cell cycle arrest by pl6INK4a. Mol Cell 6, 737-742.
Callaghan, D. A., Dong, L., Callaghan, S. M., Hou, Y. X., Dagnino, L., and Slack, R. S.
(1999). Neural precursor cells differentiating in the absence of Rb exhibit delayed
terminal mitosis and deregulated E2F 1 and 3 activity. Dev Biol 207, 257-270.
Camarda, G., Siepi, F., Pajalunga, D., Bernardini, C., Rossi, R., Montecucco, A., Meccia,
E., and Crescenzi, M. (2004). A pRb-independent mechanism preserves the postmitotic
state in terminally differentiated skeletal muscle cells. J Cell Biol 167, 417-423.
Castano, E., Kleyner, Y., and Dynlacht, B. D. (1998). Dual cyclin-binding domains are
required for p107 to function as a kinase inhibitor. Mol Cell Biol 18, 5380-5391.
Chen, C. R., Kang, Y., Siegel, P. M., and Massague, J. (2002). E2F4/5 and p107 as Smad
cofactors linking the TGFbeta receptor to c-myc repression. Cell 110, 19-32.
Chen, D., Livne-bar, I., Vanderluit, J. L., Slack, R. S., Agochiya, M., and Bremner, R.
(2004). Cell-specific effects of RB or RB/p 107 loss on retinal development implicate an
intrinsically death-resistant cell-of-origin in retinoblastoma. Cancer Cell 5, 539-551.
Chibazakura, T., McGrew, S. G., Cooper, J. A., Yoshikawa, H., and Roberts, J. M.
(2004). Regulation of cyclin-dependent kinase activity during mitotic exit and
maintenance of genome stability by p21, p27, and p107. Proc Natl Acad Sci U S A 101,
4465-4470.
Chu, W. M., Wang, Z., Roeder, R. G., and Schmid, C. W. (1997). RNA polymerase III
transcription repressed by Rb through its interactions with TFIIIB and TFIIIC2. J Biol
Chem 272, 14755-14761.
232
Classon, M., and Dyson, N. (2001). p107 and p130: versatile proteins with interesting
pockets. Exp Cell Res 264, 135-147.
Classon, M., Salama, S., Gorka, C., Mulloy, R., Braun, P., and Harlow, E. (2000).
Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control. Proc
Natl Acad Sci U S A 97, 10820-10825.
Claudio, P. P., Tonini, T., and Giordano, A. (2002). The retinoblastoma family: twins or
distant cousins? Genome Biol 3, reviews3012.
Cobrinik, D., Lee, M. H., Hannon, G., Mulligan, G., Bronson, R. T., Dyson, N., Harlow,
E., Beach, D., Weinberg, R. A., and Jacks, T. (1996). Shared role of the pRB-related
p130 and p107 proteins in limb development. Genes Dev 10, 1633-1644.
Dannenberg, J. H., van Rossum, A., Schuijff, L., and te Riele, H. (2000). Ablation of the
retinoblastoma gene family deregulates G(1) control causing immortalization and
increased cell turnover under growth-restricting conditions. Genes Dev 14, 3051-3064.
de Bruin, A., Wu, L., Saavedra, H. I., Wilson, P., Yang, Y., Rosol, T. J., Weinstein, M.,
Robinson, M. L., and Leone, G. (2003). Rb function in extraembryonic lineages
suppresses apoptosis in the CNS of Rb-deficient mice. Proc Natl Acad Sci U S A 100,
6546-6551.
Ferguson, K. L., Vanderluit, J. L., Hebert, J. M., McIntosh, W. C., Tibbo, E., MacLaurin,
J. G., Park, D. S., Wallace, V. A., Vooijs, M., McConnell, S. K., and Slack, R. S. (2002).
Telencephalon-specific Rb knockouts reveal enhanced neurogenesis, survival and
abnormal cortical development. Embo J 21, 3337-3346.
Fletcher, O., Easton, D., Anderson, K., Gilham, C., Jay, M., and Peto, J. (2004). Lifetime
risks of common cancers among retinoblastoma survivors. J Natl Cancer Inst 96, 357363.
Gad, A., Thullberg, M., Dannenberg, J. H., te Riele, H., and Stromblad, S. (2004).
Retinoblastoma susceptibility gene product (pRb) and p107 functionally separate the
requirements for serum and anchorage in the cell cycle Gi-phase. J Biol Chem 279,
13640-13644.
Gaubatz, S., Lindeman, G. J., Ishida, S., Jakoi, L., Nevins, J. R., Livingston, D. M., and
Rempel, R. E. (2000). E2F4 and E2F5 play an essential role in pocket protein-mediated
G1 control. Mol Cell 6, 729-735.
Gupta, S., Luong, M. X., Bleuming, S. A., Miele, A., Luong, M., Young, D., Knudsen, E.
S., Van Wijnen, A. J., Stein, J. L., and Stein, G. S. (2003). Tumor suppressor pRB
functions as a co-repressor of the CCAAT displacement protein (CDP/cut) to regulate
cell cycle controlled histone H4 transcription. J Cell Physiol 196, 541-556.
233
Hannan, K. M., Hannan, R. D., Smith, S. D., Jefferson, L. S., Lun, M., and Rothblum, L.
I. (2000a). Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the
interaction between UBF and SL-1. Oncogene 19, 4988-4999.
Hannan, K. M., Kennedy, B. K., Cavanaugh, A. H., Hannan, R. D., HirschlerLaszkiewicz, I., Jefferson, L. S., and Rothblum, L. I. (2000b). RNA polymerase I
transcription in confluent cells: Rb downregulates rDNA transcription during confluenceinduced cell cycle arrest. Oncogene 19, 3487-3497.
Helin, K., Harlow, E., and Fattaey, A. (1993). Inhibition of E2F-1 transactivation by
direct binding of the retinoblastoma protein. Mol Cell Biol 13, 6501-6508.
Hernando, E., Nahle, Z., Juan, G., Diaz-Rodriguez, E., Alaminos, M., Hemann, M.,
Michel, L., Nlittal, V., Gerald, W., Benezra, R., et al. (2004). Rb inactivation promotes
genomic instability by uncoupling cell cycle progression from mitotic control. Nature
430, 797-802.
Herrera, R. E., Sah, V. P., Williams, B. O., Makela, T. P., Weinberg, R. A., and Jacks, T.
(1996). Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in
Rb-deficient fibroblasts. Mol Cell Biol 16, 2402-2407.
Hirsch, H. A., Gu, L., and Henry, R. W. (2000). The retinoblastoma tumor suppressor
protein targets distinct general transcription factors to regulate RNA polymerase III gene
expression. Mol Cell Biol 20, 9182-9191.
Hirsch, H. A., Jawdekar, G. W., Lee, K. A., Gu, L., and Henry, R. W. (2004). Distinct
mechanisms for repression of RNA polymerase III transcription by the retinoblastoma
tumor suppressor protein. Mol Cell Biol 24, 5989-5999.
Huh, M. S., Parker, M. H., Scime, A., Parks, R., and Rudnicki, M. A. (2004). Rb is
required for progression through myogenic differentiation but not maintenance of
terminal differentiation. J Cell Biol 166, 865-876.
lavarone, A., King, E. R., Dai, X. M., Leone, G., Stanley, E. R., and Lasorella, A. (2004).
Retinoblastoma promotes definitive erythropoiesis by repressing Id2 in fetal liver
macrophages. Nature 432, 1040-1045.
Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, M. A., and Weinberg, R.
A. (1992). Effects of an Rb mutation in the mouse. Nature 359, 295-300.
Jori, F. P., Napolitano, M. A., Melone, M. A., Cipollaro, M., Cascino, A., Giordano, A.,
and Galderisi, U. (2004). Role of RB and RB2/P130 genes in marrow stromal stem cells
plasticity. J Cell Physiol 200, 201-212.
Jung, J., Kim, T. G., Lyons, G., Kim, H. R., and Lee, Y. (2005). Jumonji regulates
cardiomyocyte proliferation via interaction with retinoblastoma protein. J Biol Chem.
234
Knudsen, E. S., and Wang, J. Y. (1997). Dual mechanisms for the inhibition of E2F
binding to RB by cyclin-dependent kinase-mediated RB phosphorylation. Mol Cell Biol
17, 5771-5783.
Laplantine, E., Rossi, F., Sahni, M., Basilico, C., and Cobrinik, D. (2002). FGF signaling
targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest. J
Cell Biol 158, 741-750.
Lasorella, A., Iavarone, A., and Israel, M. A. (1996). Id2 specifically alters regulation of
the cell cycle by tumor suppressor proteins. Mol Cell Biol 16, 2570-2578.
Lasorella, A., Rothschild, G., Yokota, Y., Russell, R. G., and Iavarone, A. (2005). Id2
mediates tumor initiation, proliferation, and angiogenesis in Rb mutant mice. Mol Cell
Biol 25, 3563-3574.
Lee, E. Y., Cam, H., Ziebold, U., Rayman, J. B., Lees, J. A., and Dynlacht, B. D. (2002).
E2F4 loss suppresses tumorigenesis in Rb mutant mice. Cancer Cell 2, 463-472.
Lee, E. Y., Chang, C. Y., Hu, N., Wang, Y. C., Lai, C. C., Herrup, K., Lee, W. H., and
Bradley, A. (1992). Mice deficient for Rb are nonviable and show defects in neurogenesis
and haematopoiesis. Nature 359, 288-294.
Lemercier, C., Duncliffe, K., Boibessot, I., Zhang, H., Verdel, A., Angelov, D., and
Khochbin, S. (2000). Involvement of retinoblastoma protein and HBP1 in histone H1(0)
gene expression. Mol Cell Biol 20, 6627-6637.
Leone, G., Nuckolls, F., Ishida, S., Adams, M., Sears, R., Jakoi, L., Miron, A., and
Nevins, J. R. (2000). Identification of a novel E2F3 product suggests a mechanism for
determining specificity of repression by Rb proteins. Mol Cell Biol 20, 3626-3632.
Lipinski, M. M., Macleod, K. F., Williams, B. O., Mullaney, T. L., Crowley, D., and
Jacks, T. (2001). Cell-autonomous and non-cell-autonomous functions of the Rb tumor
suppressor in developing central nervous system. Embo J 20, 3402-3413.
MacLellan, W. R., Garcia, A., Oh, H., Frenkel, P., Jordan, M. C., Roos, K. P., and
Schneider, M. D. (2005). Overlapping roles of pocket proteins in the myocardium are
unmasked by germ line deletion of p130 plus heart-specific deletion of Rb. Mol Cell Biol
25, 2486-2497.
MacPherson, D., Sage, J., Crowley, D., Trumpp, A., Bronson, R. T., and Jacks, T. (2003).
Conditional mutation of Rb causes cell cycle defects without apoptosis in the central
nervous system. Mol Cell Biol 23, 1044-1053.
MacPherson, D., Sage, J., Kim, T., Ho, D., McLaughlin, M. E., and Jacks, T. (2004). Cell
type-specific effects of Rb deletion in the murine retina. Genes Dev 18, 1681-1694.
235
Marino, S., Hoogervoorst, D., Brandner, S., and Berns, A. (2003). Rb and p107 are
required for normal cerebellar development and granule cell survival but not for Purkinje
cell persistence. Development 130, 3359-3368.
Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J., and Berns, A. (2000). Induction
of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the
external granular layer cells of the cerebellum. Genes Dev 14, 994-1004.
Miura, T., Mattson, M. P., and Rao, M. S. (2004). Cellular lifespan and senescence
signaling in embryonic stem cells. Aging Cell 3, 333-343.
Morris, E. J., and Dyson, N. J. (2001). Retinoblastoma protein partners. Adv Cancer Res
82, 1-54.
Novitch, B. G., Mulligan, G. J., Jacks, T., and Lassar, A. B. (1996). Skeletal muscle cells
lacking the retinoblastoma protein display defects in muscle gene expression and
accumulate in S and G2 phases of the cell cycle. J Cell Biol 135, 441-456.
Novitch, B. G., Spicer, D. B., Kim, P. S., Cheung, W. L., and Lassar, A. B. (1999). pRb
is required for MEF2-dependent gene expression as well as cell-cycle arrest during
skeletal muscle differentiation. Curr Biol 9, 449-459.
Paggi, M. G., and Giordano, A. (2001). Who is the boss in the retinoblastoma family?
The point of view of Rb2/p130, the little brother. Cancer Res 61, 4651-4654.
Papadimou, E., Menard, C., Grey, C., and Puceat, M. (2005). Interplay between the
retinoblastoma protein and LEK1 specifies stem cells toward the cardiac lineage. Embo J.
Rayman, J. B., Takahashi, Y., Indjeian, V. B., Dannenberg, J. H., Catchpole, S., Watson,
R. J., te Riele, H., and Dynlacht, B. D. (2002). E2F mediates cell cycle-dependent
transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor
complex. Genes Dev 16, 933-947.
Rodier, G., Makris, C., Coulombe, P., Scime, A., Nakayama, K., Nakayama, K. I., and
Meloche, S. (2005). p107 inhibits G1 to S phase progression by down-regulating
expression of the F-box protein Skp2. J Cell Biol 168, 55-66.
Sage, J., Miller, A. L., Perez-Mancera, P. A., Wysocki, J. M., and Jacks, T. (2003). Acute
mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature 424,
223-228.
Sage, J., Mulligan, G. J., Attardi, L. D., Miller, A., Chen, S., Williams, B., Theodorou, E.,
and Jacks, T. (2000). Targeted disruption of the three Rb-related genes leads to loss of
G(1) control and immortalization. Genes Dev 14, 3037-3050.
Savatier, P., Huang, S., Szekely, L., Wiman, K. G., and Samarut, J. (1994). Contrasting
patterns of retinoblastoma protein expression in mouse embryonic stem cells and
embryonic fibroblasts. Oncogene 9, 809-818.
236
Schmitz, N. M., Leibundgut, K., and Hirt, A. (2004). MCM4 shares homology to a
replication/DNA-binding domain in CTF and is contacted by pRb. Biochem Biophys Res
Commun 317, 779-786.
Stead, E., White, J., Faast, R., Conn, S., Goldstone, S., Rathjen, J., Dhingra, U., Rathjen,
P., Walker, D., and Dalton, S. (2002). Pluripotent cell division cycles are driven by
ectopic Cdk2, cyclin A/E and E2F activities. Oncogene 21, 8320-8333.
Sullivan, C. S., Baker, A. E., and Pipas, J. M. (2004). Simian virus 40 infection disrupts
p130-E2F and p107-E2F complexes but does not perturb pRb-E2F complexes. Virology
320, 218-228.
Takahashi, C., Bronson, R. T., Socolovsky, M., Contreras, B., Lee, K. Y., Jacks, T.,
Noda, M., Kucherlapati, R., and Ewen, M. E. (2003). Rb and N-ras function together to
control differentiation in the mouse. Mol Cell Biol 23, 5256-5268.
Tedesco, D., Lukas, J., and Reed, S. I. (2002). The pRb-related protein p130 is regulated
by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2).
Genes Dev 16, 2946-2957.
Thomas, D. M., Carty, S. A., Piscopo, D. M., Lee, J. S., Wang, W. F., Forrester, W. C.,
and Hinds, P. W. (2001). The retinoblastoma protein acts as a transcriptional coactivator
required for osteogenic differentiation. Mol Cell 8, 303-316.
Thomas, D. M., Johnson, S. A., Sims, N. A., Trivett, M. K., Slavin, J. L., Rubin, B. P.,
Waring, P., McArthur, G. A., Walkley, C. R., Holloway, A. J., et al. (2004). Terminal
osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.
J Cell Biol 167, 925-934.
Trimarchi, J. M., and Lees, J. A. (2002). Sibling rivalry in the E2F family. Nat Rev Mol
Cell Biol 3, 11-20.
Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L., and Jacks, T. (1998).
Mutation of E2f--1 suppresses apoptosis and inappropriate S phase entry and extends
survival of Rb-deficient mouse embryos. Mol Cell 2, 293-304.
Voit, R., Schafer, K., and Grummt, I. (1997). Mechanism of repression of RNA
polymerase I transcription by the retinoblastoma protein. Mol Cell Biol 17, 4230-4237.
White, J., Stead, E., Faast, R., Conn, S., Cartwright, P., and Dalton, S. (2005).
Developmental activation of the Rb-E2F pathway and establishment of cell cycleregulated cyclin-dependent kinase activity during embryonic stem cell differentiation.
Mol Biol Cell 16, 2018-2027.
White, R. J. (2004). RNA polymerase III transcription and cancer. Oncogene 23, 32083216.
237
White, R. J. (2005). RNA polymerases I and III, growth control and cancer. Nat Rev Mol
Cell Biol 6, 69-78.
Williams, B. O., Schmitt, E. M., Remington, L., Bronson, R. T., Albert, D. M., Weinberg,
R. A., and Jacks, T. (1994). Extensive contribution of Rb-deficient cells to adult chimeric
mice with limited histopathological consequences. Embo J 13, 4251-4259.
Woo, M. S., Sanchez, I., and Dynlacht, B. D. (1997). p130 and p107 use a conserved
domain to inhibit cellular cyclin-dependent kinase activity. Mol Cell Biol 17, 3566-3579.
Wu, L., de Bruin, A., Saavedra, H. I., Starovic, M., Trimboli, A., Yang, Y., Opavska, J.,
Wilson, P., Thompson, J. C., Ostrowski, M. C., et al. (2003). Extra-embryonic function
of Rb is essential for embryonic development and viability. Nature 421, 942-947.
Xiao, A., Wu, H., Pandolfi, P. P., Louis, D. N., and Van Dyke, T. (2002). Astrocyte
inactivation of the pRb pathway predisposes mice to malignant astrocytoma development
that is accelerated by PTEN mutation. Cancer Cell 1, 157-168.
Yamasaki, L., Bronson, R., Williams, B. O., Dyson, N. J., Harlow, E., and Jacks, T.
(1998). Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rbl(+/-)mice.
Nat Genet 18, 360-364.
Young, A. P., and Longmore, G. D. (2004). Differences in stability of repressor
complexes at promoters underlie distinct roles for Rb family members. Oncogene 23,
814-823.
Zhu, L., Harlow, E., and Dynlacht, B. D. (1995). p107 uses a p21CIP1-related domain to
bind cyclin/cdk2 and regulate interactions with E2F. Genes Dev 9, 1740-1752.
Ziebold, U., Lee, E. Y., Bronson, R. T., and Lees, J. A. (2003). E2F3 loss has opposing
effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but
metastasis of medullary thyroid carcinomas. Mol Cell Biol 23, 6542-6552.
Ziebold, U., Reza, T., Caron, A., and Lees, J. A. (2001). E2F3 contributes both to the
inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev
15, 386-391.
238