GCNF AS A GROWTH REGULATOR IN NORMAL AND NEOPLASTIC CELLS
A Thesis by
Sowmya Srikanthan
Bachelor of Science, ADU, India, 2001
Master of Science, MKU, India, 2003
Submitted to the College of Liberal Arts and Sciences
and the faculty of the Graduate School of
Wichita State University
in partial fulfillment of the requirements for the degree of
Master of Science
December 2006
GCNF AS A GROWTH REGULATOR IN NORMAL AND NEOPLASTIC CELLS
I have examined the final copy of this thesis for form and content and recommend that it
be accepted in partial fulfillment of the requirements for the degree of Master of Science
with a major in Biological Sciences
________________________________________
Jeffrey V. May PhD, Committee Chair
We have read this thesis and recommend its acceptance:
_____________________________________________
William J. Hendry III PhD, Committee Member
____________________________________________
Dr. David McDonald PhD, Committee Member
___________________________________________
Dr. John Carter PhD, Committee Member
ii
ACKNOWLEDGEMENTS
I sincerely thank my advisor Dr. Jeffrey May. His support and guidance have
always been constant and without this, nothing would have been possible; his wisdom
and passion for science and his vivid ideas have always been so inspiring. My
appreciation extends to my committee members Dr. William Hendry and Dr. David
McDonald and Dr. John Carter. Thank you for your guidance, assistance, and insightful
comments. A special thank you goes to Dr. Austin Cooney and Dr. Li Jia for their
technical contributions to this project. Also I extend my sincere thanks to Jessica Bowser
for all the help and support.
I would like to thank Isabel Hendry, Dr. Yun-Hwa Mau, Ellie Skokan, Maria
Russell, and numerous individuals, too many to name, who have given me helpful advice
either scientifically or personally over the course of my studies.
I extend a sincere appreciation to my family for always encouraging me and
supporting me throughout, without which this would not have been possible. Finally
thanks to my beloved Husband for his encouragement and support.
iii
ABSTRACT
Germ cell nuclear factor (GCNF/NCNF/RTR, NR6A1) is an orphan member of
the nuclear receptor superfamily that exhibits repressive transcriptional activity and is
expressed in the embryonic stem cells, embryonic carcinoma cells and reportedly
relegated to the germ line in adults. We have demonstrated that GCNF mRNA and
protein are expressed in four of the four ovarian cancer cell lines tested: two
adenocarcinomas, OVCAR-3 and TOV-112; one clear-cell carcinoma, ES-2; and one,
teratocarcinoma, PA-1. The four cell lines exhibited varied morphologies in culture and
exhibited markedly different growth rates in vitro. Immunoblot analysis of GCNF protein
expression by the cell lines revealed several GCNF antibody-specific bands, the sizes of
which were consistent with the reported homodimeric and multimeric associations of
GCNF. In addition, GCNF mRNA was highly expressed in mink lung epithelial cells
(MLEC), a rapidly proliferating, non-transformed epithelial cell line. Taken together,
these findings suggest a relationship between GCNF and cell proliferation. GCNF gene
silencing was employed to investigate this relationship in ovarian cancer cells maintained
in vitro with the help of siRNA synthesized by Santa Cruz Biotechnology. The results
showed that GCNF siRNA treatment decreased the monolayer cell content by 45.6% and
36.2% of ES-2 and TOV-112 cells, relative to untreated and control siRNA-treated cells.
Consistent with the observed decline in cell proliferation, GCNF siRNA treatment
decreased the cellular content of GCNF mRNA by 37.6% in TOV-112 cells. These
preliminary results implicate GCNF as a here-to-fore unrecognized and potentially
important growth regulator in ovarian cancer cells.
iv
Another experiment was also undertaken to determine the effects of growth
suppression upon GCNF expression using a non-transformed cell line, the mink lung
epithelial cells (MLEC). These non-transformed cells (MLEC) express GCNF and are
both contact inhibited and exquisitely sensitive to TGF-β1, which markedly inhibits
proliferation. MLEC were cultured in the presence and absence of TGF-β1 (1 and 5ng)
which showed a dose dependent decrease in both GCNF mRNA expression and cell
proliferation which suggests a relationship between growth and GCNF.
To demonstrate potential regulation of GCNF, several of the cell lines were
treated with all trans-retinoic acid during moderate-term, monolayer culture. Retinoic
acid increased the expression of GCNF mRNA within 48 hours of exposure in ES-2,
OVCAR and TOV-112 cells. While retinoic acid increased GCNF mRNA expression it
did not decrease Oct-4 mRNA expression. GCNF has been reported to repress several
key genes including Oct-4, involved in the maintenance of stem cells and GDF-9, a TGFβ family member. In the ovarian cancer cell lines expressing GCNF mRNA, both Oct-4
and GDF-9 mRNAs were detected despite GCNF expression, suggesting an impaired
function of GCNF.
A conserved domain search was also performed for both the domains for GCNF
(the DNA binding domain and the ligand binding domain) and also a multiple alignment
for the highly conserved regions which displayed 3D-structures for both the domains.
The DNA binding domain of GCNF shares a 3D homology with 1HLZ, which is the
Homo sapiens chain A crystal structure of orphan nuclear receptor Rev-Erb (Alpha),
named because it is encoded on the opposite strand of the T3R .
v
The ligand binding domain shares a high degree of structural homology (3D)
with 1HG4, chain A ultraspiracle ligand binding domain from Drosophila. As USP has
RXR as its mammalian homologue, GCNF (the ligand binding domain) shares a higher
degree of homology with RXR than with any other nuclear receptor.
Our results altogether demonstrate the novel expression of GCNF in ovarian
cancer cells and suggest that at least some of the normal regulatory actions of GCNF may
be impaired or altered. To what extent this contributes to the neoplastic basis of the
ovarian cancer cells is unclear, but the finding of GCNF expression opens new avenues
for future investigation and perhaps a new therapeutical target.
vi
TABLE OF CONTENTS
Chapter
I.
Page
INTRODUCTION
Nuclear Hormone Receptors
Orphan Nuclear Receptors
Mechanism of Action
Domain Studies
DNA Binding Domain
Ligand Binding Domain
Specific Aims
II.
1
4
5
6
7
9
11
MATERIALS AND METHODS
Growth and Maintenance of Ovarian Cancer Cells
Determination of Cell Growth Rates
GCNF, Oct-4, GDF-9, and FSH Receptor Oligonucleotide
Primer Formulation
RNA Isolation and RT/PCR
DNA Sequencing
Immunoblot Detection of GCNF Protein
Impact of Retinoic Acid upon Ovarian Cancer Cell
GCNF mRNA Expression
siRNA transfection
A Conserved Domain Database and Search Service, v2.09
Cn3D 4.1
Statistical Analysis
III.
13
14
14
16
17
17
19
19
21
21
22
RESULTS
Expression of GCNF mRNA in the Normal Ovary
Characteristics of Ovarian Cancer Cell Lines Utilized
Expression of GCNF mRNA in Ovarian Cancer Cell Lines
Expression of GCNF protein by ovarian cancer cell Lines
Expression of GDF-9 and Oct-4 by ovarian cancer cell lines
Impact of retinoic acid upon GCNF expression
GCNF gene knock down (silencing) to determine the
effects of GCNF on Growth
Experiment to determine the effects of Growth
suppression on GCNF expression
23
23
24
25
25
26
27
28
IV.
DISCUSSION
29
V.
REFERENCES
37
VI.
APPENDICES
48
vii
LIST OF ABBREVIATIONS/NOMENCLATURE
AF
Activation Function
atRA
All-trans retinoic acid
Bax
bcl-2 associated-x gene
BLAST
Basic Local Alignment Search Tool
bcl-2
B-cell leukemia/lymphoma-2
BMP-15
Bone Morphogenetic Protein-15
bp
Nucleotide Base Pair
C
Celsius
cc
Cubic Centimeters
CDD
Conserved Domain Database
CO2
Carbon Dioxide
COUP-TF
(chicken ovalbumin upstream promoter transcription factor
CTE
Carboxy terminal extension
DBD
DNA binding domain
DEPC
Diethylprocarbonate
DMSO
Dimethyl sulphoxide
DNA
Deoxyribonucleic Acid
EDTA
Ethylenediaminetetraacetic Acid
ERα
Estrogen Receptor α
ERβ
Estrogen Receptor β
EtOH
Ethanol
viii
LIST OF ABBREVIATIONS/NOMENCLATURE (continued)
FCS
Fetal Calf Serum
FSH
Follicle Stimulating Hormone
FSHR
Follicle Stimulating Hormone Receptor
GCNF
Germ cell nuclear factor
GDF-9
Growth Differentiation Factor-9
HAT
Histone Acetyl Transferase
HDAC
Histone Deacetylase
HRE
Hormone Response Element
IGF-1
Insulin-like Growth Factor 1
KDA
Kilodalton
Kg
Kilogram
LBD
Ligand Binding Domain
LH
Lutenizing Hormone
M
Molar
ml
Milliliter
MLEC
Mink Lung Epithelial Cells
MMDB
Molecular Modeling Database
mRNA
Messenger Ribonucleic Acid
NCBI
National Center for Biotechnology Information
ng
Nanogram
nm
Nanometer
ix
LIST OF ABBREVIATIONS/NOMENCLATURE (continued)
Oct-4
Octamer-4
PBS
Phosphate-Buffered Saline
PCR
Polymerase Chain Reaction
PGC
Primordial Germ Cells
PDB
Protein Data Bank
pmol
Picomole
POF
Polyovular Follicles
RAR
Retinoic Acid Receptor
Rev-Erb
opposite strand of the T3R
RNA
Ribonucleic Acid
RORRAR
Related Orphan Receptor
RPM
Revolutions per Minute
RT
Reverse Transcription
RT-PCR
Reverse Transcription-Polymerase Chain Reaction
RXR
Retinoid X Receptor
SDS
Sodium Dodecylsulfate
SF-1
Steroidogenic Factor 1
siRNA
Small interference RNA
TBS
Tris-Buffered Saline
TGF-β
Transforming Growth Factor β
µg
Microgram
µl
Microliter
x
LIST OF ABBREVIATIONS/NOMENCLATURE (continued)
USP
Ultraspiracle
UV
Ultraviolet
v/v
Volume to Volume
WT1
Wilms Tumor Suppressor 1
w/v
Weight to Volume
xi
INTRODUCTION
Nuclear Hormone Receptors
Nuclear receptors control gene expression in multicellular organisms in response
to a wide range of developmental, physiological and environmental cues [1]. Nuclear
receptor activity can be regulated by at least three different mechanisms: a) When a small
lipophilic ligand binds to the receptor or its partner in heterodimer complexes; b) When
there is a covalent modification, usually in the form of phosphorylation, controlled by
events at the cellular membrane or during the cell cycle; and c) During protein-protein
interactions, mostly through contacts with other transcription factors including nuclear
receptors themselves. All three mechanisms can either work individually or in concert
with each other to modulate a specific signal [1]. Prototypical NRs share a common
structural organization with a variable modulator domain, a conserved DNA-binding
domain (DBD) and a C-terminal (Cter) ligand-binding domain (LBD). From a regulatory
point of view, the LBD is the most important domain for most nuclear receptors,
containing both the ligand-binding pocket and the ligand-dependent transactivation
function. It possesses interaction surfaces for the homo- or heterodimeric partner, corepressors, co-activators and other bridging factors [2].
The modulator domain normally contains a transcriptional activation function,
referred to as AF-1. Estrogen and progesterone receptor studies have clearly
demonstrated that the modulator domains possess promoter and cell context-dependent
functions suggesting that the amino-terminal region of nuclear receptors may interact
with cell-specific cofactors [3].
1
The modulator domain, which is also referred to as the A/B domain, is highly
variable both in terms of length and primary amino acid sequence. Most of the nuclear
receptors use, different promoters, alternative splicing and distinct translational start sites
to generate multiple modulator domains, leading to the expression of many receptor
isoforms from a single gene. This phenomenon is best characterized by the family of
retinoic acid receptors for which three genes produce at least eight receptors with similar
DNA- and ligand-binding properties but distinct biological functions [3, 4].
The DBD of nuclear receptors is the most conserved domain which is composed
of two zinc finger modules encoded by 66–70 amino acid residues and a carboxyterminal extension (CTE) that spans approximately 25 residues. The DNA binding
domains of the nuclear receptors bind DNA as monomers, homodimers, and heterodimers
[5]. Though retinoid X receptor (RXR) is a common partner in most of the heterodimeric
complexes, alternative heterodimeric interactions between nuclear receptors have also
been reported and may be of physiological significance [6,7]. Nuclear receptor DNA
recognition sites, also referred to as hormone response elements (HREs), contain one or
two consensus core half-site sequences. For dimeric HREs, the half-sites can be
configured as inverted or direct repeats. Estrogen receptors and other nuclear receptors
bind to the half-site consensus sequence AGGTCA while the steroid receptors recognize
the half-site consensus sequence AGAACA. For monomeric HREs, a single half-site is
preceded by a 5'-flanking A/T-rich sequence. Half-site sequences can deviate to a large
extent from the consensus sequences, especially for dimeric HREs in which a single
conserved half-site is usually sufficient to provide high-affinity binding to the homo- or
heterodimer complexes. Natural HREs rarely contain two perfect consensus half-sites [7].
2
The DBD has been further divided into sub domains involved in direct
recognition of the core half-site sequences (P-box) and dimerization determinants (D- and
DR-boxes) [8, 9]. In addition, the crystal structure of the RXR -thyroid hormone (T3)
receptor ß (T3R ß) DBD complex and computer modeling have shown that each
heterodimeric complex may utilize partner-specific dimerization determinants [10]. The
CTE provides both protein-DNA interfaces and protein-protein interfaces [11, 12].
Finally, because of the asymmetric nature of direct repeat HREs, RXR and its partner
bind DNA in a fixed orientation. In vitamin D receptor (VDR), T3R and all-trans-retinoic
acid (atRA) receptor (RAR)-RXR heterodimer complexes, RXR occupies the upstream
half-site and its partner the downstream half-site [13, 14]. The site occupied by a receptor
on a direct repeat HRE might regulate its ability to recognize its ligand [15].
The main function of the hinge region is to serve as a hinge between the DBD and
LBD. It is also highly variable in both length and primary sequence. And the hinge region
is very flexible to let the DBD rotate 180° to allow some receptors to bind as dimers to
both direct and inverted HREs. Recent studies have also demonstrated that the hinge
region may serve as a docking site for co-repressor proteins [16].
The LBD is a multifunctional domain that is associated with, ligand binding,
dimerization, interaction with heat shock proteins, nuclear localization, and
transactivation functions [16]. Nuclear receptor LBDs are defined by a signature motif
overlapping with helix 4, although quite variable in primary sequence. In addition,
ligand-dependent transactivation is dependent on the presence of a highly conserved
motif, referred to as activation function-2 (AF-2), localized at the carboxy-terminal end
of the LBD. X-ray crystallographic experiments suggest that LBDs have similar
3
structures: they are formed by the folding of 11–13 -helices into three layers that bury
the ligand-binding site within the core of the LBD [17,18]. Ligand-dependent
transactivation involves the recruitment of co-activators, a process in which the AF-2
plays an obligatory role. Comparison of holo and apo-LBD structures has led to the
mouse trap model in which ligand binding creates a conformational change in the LBD
allowing co-activators to bind [19]. In this model, the AF-2 motif folds back against the
core LBD upon ligand binding, closing the ligand-binding pocket and forming a novel
interface involving residues from the AF-2 itself and at least three other helices [20].
While transcriptionally competent interfaces are induced by receptor agonists, binding of
antagonists to the LBD leads to the formation of a non-functional interface preventing
interaction between the nuclear receptor and coactivator proteins [21].
Orphan Nuclear Receptors
Orphan nuclear receptors are defined as gene products that embody structural
features of nuclear receptors that were identified without any prior knowledge of their
association with a putative ligand. Using this definition, orphan nuclear receptors remain
in this category even after the subsequent identification of specific ligands [22].
GCNF was first described as a novel orphan receptor in mouse germ cells [23]. It
is expressed in growing spermatids and oocytes, as well as in neuronally differentiating
EC cells [24, 25]. With regard to neurogenesis, GCNF shows a spatially and temporally
regulated expression pattern in the developing nervous system of the embryo and was
found in postmitotic neuronal cells of the adult mouse brain [26]. The protein is a
sequence-specific repressor of transcription that binds as a homodimer to directly
4
repeated consensus motifs PuGGTCA with zero spacing (DR0) or an extended half site
[27]. Interaction studies suggested that repression involves the recruitment of nuclear corepressors, which may be modulated by the RTR-associated protein (RAP) 80 [28].
Known target genes are the protamine genes 1 and 2, which are expressed in round
spermatids, the genes BMP-15 (Bone Morphogenetic Protein) and GDF-9 (Growth
Differentiation Factor) that are up regulated in females at diestrus, and the OCT-4, a
master regulatory gene involved in the maintenance of pluripotency [29, 30]. Gene
targeting approaches showed that GCNF is required for the maintenance of
somitogenesis, normal anterior-posterior development, embryonic survival, and the
restriction of Oct4 expression to the germ line [31, 32]. Recently, the function of GCNF
in the differentiation of oocytes and female fertility has been unraveled via by-passing
embryonal lethality with an oocyte-specific knockout [33].
Mechanism of Action
Nuclear receptors are constitutively nuclear and often bound to DNA in the
absence of their ligand. It is also now widely recognized that in the absence of ligands,
many nuclear receptors can act as strong repressors of gene expression [34]. Nuclear
receptors have been shown to associate with various components of the general
transcription machinery, co-repressors, co-activators, and the co-integrator CBP (CREBbinding protein)/p300 [35]. Co-repressor proteins may function by recruiting histone
deacetylases, which keeps the chromatin in a repressive state [36]. Upon ligand binding,
the repressor complex dissociates from the receptor, which is then free to interact with
the coactivator complex. The receptor-coactivator complex may contain one or more
5
coactivators, including an RNA coactivator referred to as SRA [37], p/CAF (p 300/CBPassociated factor), CBP/p300, and other uncharacterized components. SRC-1, p/CAF, and
CBP have been shown to possess intrinsic histone acetylase activity leading to a
derepression of the chromatin structure [38]. Taken together, these results portray a new
model for transcriptional regulation by nuclear receptors that includes three chromatin
states: 1) normal chromatin that displays basal levels of histone acetylation and
transcription in the absence of a receptor; 2) repressive chromatin with deacetylated
histones and no transcription in the presence of the unliganded receptor; and 3) active
chromatin with high levels of histone acetylation and transcription in the presence of
liganded receptor [37, 38]. However, chromatin disruption alone is not sufficient for
transcriptional activation, indicating that additional interactions between nuclear
receptors and the general transcription machinery are required to regulate gene
expression [38].
Domain Studies
The 3D crystal structure for GCNF was not presented in the PDB (Protein Data
Bank) [39]. Thus we decided look for its structure through homology modeling. First, a
conserved domain search was done for both the domains for GCNF (the DNA binding
domain and the ligand binding domain) and then a multiple alignment for the highly
conserved domains using CDD (Conserved Domain Database) [40]. Cn3D (a
visualization tool) was used to display the 3D structure for the most conserved domains
[41]. The DNA binding domain of GCNF shares a 3D homology with 1HLZ, which is the
6
Homo sapiens chain A crystal structure of orphan nuclear receptor Rev-Erb (Alpha),
named because it is encoded on the opposite strand of the T3Rα [42].
The ligand binding domain shares a high degree of structural homology (3D) with
1HG4, chain A ultraspiracle ligand binding domain from Drosophila [43]. As USP has
RXR as its mammalian homologue, GCNF (the ligand binding domain) shares a higher
degree of homology with RXR than with any other nuclear receptor. Though we have yet
to uncover any GCNF ligand, due to its 3D structural homologies with ultraspiracle and
Rev-Erb alpha, GCNF may have a wide variety of targets and possibilities for ligand
binding.
DNA Binding Domain
Rev-Erb: Singular members of the superfamily
The original and the first member of this family, the Rev-Erb gene, was so
named because it is encoded on the opposite strand of the T3R [44]. The Rev-Erbß
gene, which was then simultaneously cloned by several groups by homology screening
[45], is also closely linked to the T3Rß gene but apparently not encoded by an
overlapping locus. It is also a strong repressor of N-Myc expression suggesting an
important role in the control of cell proliferation and organ physiology [46].
Studies using the C2C12 myoblasts differentiation model have indirectly
implicated Rev-Erb and -ß as negative regulators of myogenesis [47]. However, the
observations that both putative receptors are widely expressed during development and in
adult tissues [48] and that Rev-Erbß is a strong repressor of N-Myc expression [49, 50]
suggest that these two proteins may play a more general role in the control of cell
7
proliferation and organ physiology. Finally, it is interesting to note that the Rev-Erb
homolog in C. elegans, referred to as SEX-1, determines sex in nematodes by repressing
the transcription of the sex determining gene xol-1 [51]. Given the tissue distribution of
Rev-Erb transcripts and their chromosomal localization, it is unlikely that the two RevErbs are implicated in sex determination in mammals. However, in contrast to Rev-Erbs,
SEX-1 possesses an AF-2 domain, suggesting that the ancestral Rev-Erb gene product
may have been responsive to a ligand. Based on these observations, it may be possible to
find a ligand for members of the Rev-Erb family [52].
Rev-Erbs can bind DNA with affinity as both monomers and homodimers [53,
54]. The Rev-Erb monomeric consensus site is GAATGTAGGTCA in which the T at
position -1 and A at -4 relative to the AGGTCA are essential for high-affinity binding
[55]. The Rev-Erb homodimeric site is a DR-2, but unlike RAR-RXR, Rev-Erb binding
requires the monomeric 5'-flanking A/T-rich sequence located upstream of the first halfsite [56].
X-Ray structure analysis of the Rev-Erb DBD bound to a DR-2 HRE proved the
significance of the role played by the CTE in the recognition of 5'-flanking A/T-rich
sequence by nuclear receptors and revealed an additional role for the CTE in establishing
productive protein-protein contacts in the homodimer complex [57]. The Rev-Erb LBDs
also lack an AF-2 domain, and this feature especially may be linked to the observation
that Rev-Erbs are constitutive repressors of gene transcription [58, 59]. Transcriptional
repression by Rev-Erbs is mediated through direct interactions with the nuclear receptor
corepressors N-CoR, SMRT (silencing mediator for RAR and thyroid hormone receptor),
and SUN-CoR [60]. Stoichiometric studies have led to the suggestion that Rev-Erb can
8
repress transcription only via DR-2 HREs, but not from monomeric sites, as binding of
N-CoR appears to require two receptor carboxyl termini [61]. However, both Rev-Erb
and -ß were shown to repress basal transcription via monomeric sites contained within
the natural regulatory sequences of the ApoA-1 and N-Myc genes, respectively [62].
Ligand Binding Domain
RXRs
RXR was first cloned as a result of its homology with the RAR DBD [63].
Three RXR gene products referred to as RXR , -ß, and - were characterized in
mammals [64]. Ultraspiracle, a Drosophila homolog was also identified [65]. RXRs are
ubiquitously expressed as a family, although individual RXR genes display unique but
overlapping pattern of expression during development and in adult tissues [66, 67].
RXR could be activated by supraphysiological doses of atRA, indicating that the
natural ligand for RXR might be a metabolite of atRA [68]. Starting with this idea, two
groups independently found 9-cis-retinoic acid (9cRA) as the RXR ligand [69]. The
identification of 9cRA as the natural RXR ligand was the first demonstration of reverse
endocrinology, where the discovery of a receptor leads to the identification of a novel
hormone. As 9cRA was also found to be a high-affinity ligand for RAR [70], a search for
natural and synthetic RXR-specific ligands began. Two noncyclic terpenoids, methoprene
acid and phytanic acid, were found to bind and activate RXRs in a specific manner [71,
72]. Phytanic acid is a natural chlorophyll metabolite present in normal human diet, while
methoprene acid is an environmental contaminant. While both ligands have the ability to
regulate or disrupt natural RXR responses, the physiological significance of these
9
findings remains to be proven, especially in view of the relative low affinities of these
compounds for RXRs.
As indicated above, RXRs participate in a variety of hormone response systems
via their association with other nuclear receptors as heterodimeric partners. There are two
types of RXR heterodimeric complexes. Non-permissive heterodimers are the ones which
can be activated only by the partner’s ligand [73, 74] and permissive heterodimers are the
ones which can be activated either by RXR or by the partner’s ligand [75]. Nonpermissive heterodimers include RAR/RXR, T3R/RXR, and VDR/RXR, although RXRs
are only completely silent in the T3R/RXR and VDR/RXR complexes. Both RAR and
RXR ligands can activate the RAR/RXR complex [76]; but, RXR ligands are effective
only in the presence of a RAR ligand [77]. The potential of each heterodimeric complex
to allow RXR ligand binding may be explained in part by distinct intermolecular
interactions between the RXR AF-2 domain and the coactivator-docking site of its
partner [78]. Since RXR-selective compounds can elicit a response from both retinoidand non-retinoid-related pathways, the term rexinoids is now being used to differentiate
RXR-specific activators from other vitamin A derivatives and synthetic analogs acting as
RAR ligands. The development of rexinoids has considerably extended the therapeutic
repertoire of vitamin A derivatives. Rexinoids have recently been shown to inhibit the
growth of atRA-resistant human breast cancer cells [79], act as chemopreventive agents
and even cause regression of mammary carcinoma in the rat [80], and sensitize diabetic
and obese mice to insulin [81]. Because RXRs play multiple roles in nuclear receptor
signaling, it has been difficult to assess the precise contribution of RXR-selective
pathways in developmental and physiological processes [81].
10
SPECIFIC AIMS
Our initial investigation was with regard to looking at the expression of GCNF in the
developing hamster ovary as GCNF was specific to germ cells in adults and to validate
that the PCR product by sequencing. After this investigation, the quest for looking at the
GCNF expression began, initially in the germ cell tumor line and later in many other
epithelial cancer cell lines. The specific aims of this project is as follows,
•
To investigate the mRNA expression in several ovarian cancer cell lines (ES-2,
TOV-112, PA-1, OVCAR and a non-transformed cell line, MLEC) by RT-PCR
and also validate the PCR products by sequencing.
•
To investigate the GCNF protein expression in all the cancer cell lines OVCAR 3,
ES-2, TOV-112D, CRL-1572 and MLEC. Detergent solubilized cell lysates and
tissues will be subjected to immunoblot analysis using GCNF antibodies provided
by our collaborator, Dr. Austin Cooney (Baylor College of medicine).
•
To investigate the GDF-9 and Oct-4 mRNA expression in the ovarian cancer cell
lines by RT-PCR as GCNF is reported to suppress both GDF-9 and Oct-4
expression.
•
To also determine the impact of retinoic acid upon GCNF expression based on the
fact that GCNF is up regulated during retinoic acid induced differentiation in
embryonic stem cells and embryonic carcinoma cells, while the pluripotency
11
genes like Oct-4 are down regulated at this time. This experiment is to verify
whether GCNF is acting normally in the cancer cell lines upon retinoic acid
treatment.
•
To investigate the impact on cell proliferation when GCNF gene is knocked down
using 20-25bp siRNA in all four ovarian cancer cell lines and the cell counts are
determined using the Coulter Counter. The gene knock down should later be
verified using RT/PCR. This is to determine whether suppression of GCNF
expression affects proliferation.
•
To culture the mink lung epithelial cells which are both contact inhibited and
exquisitively sensitive to TGF-beta1 both in the presence and absence of TGFbeta 1, look at GCNF mRNA expression using RT/PCR. This is to determine
whether growth inhibition affects GCNF expression.
•
To investigate the structural components (both the DNA and the Ligand binding
domains) of GCNF.
12
MATERIALS AND METHODS
Growth and Maintenance of Ovarian Cancer Cells
Ovarian cancer cell lines were obtained as frozen samples from the American
Type Culture Collection (ATCC, Bethesda, MD). The cell lines included: TOV-112D
(CRL-11731), a primary malignant adenocarcinoma; NIH: OVCAR-3 (HTB-161), an
epithelial adenocarcinoma; ES-2 (CRL-1978), a clear cell carcinoma; and PA-1 (CRL1572), a metastatic, ovarian teratocarcinoma. Upon arrival, the cells were thawed rapidly
at 37C and immediately placed into a 75 cm2 containing 30 ml of the appropriate culture
medium for each cell type as defined in ATCC protocols. Cells were cultured in a CO2
water-jacketed incubator until near confluence whereupon the cells were detached using
trypsin/EDTA and diluted with fresh cell-specific culture medium. One milliliter of the
resuspended cells was placed in the respective medium containing 5% DMSO and then
transferred to the cryotubes. The tubes were placed into a styrofoam cooler and placed
into a –80C freezer overnight. The next day, the cryotubes were submerged into liquid
nitrogen and stored there until utilized. When needed, vials of cells were thawed and
cultured as above to replenish the supply of cells. For experimentation, -195C vials of
cells were thawed rapidly by submersion into 37C water. The cells were placed into 75
cm2 flasks containing 25 ml of appropriate medium and cultured for 2-3 days. PA-1
ovarian teratocarcinoma cells were cultured in FD medium (Ham’s F-12: DMEM, 1:1)
supplemented with 10% fetal calf serum. ES-2 ovarian adenocarcinoma cells were
cultured in McCoy’s 5A medium supplemented with 10% fetal calf serum. TOV-112D
ovarian carcinoma cells were cultured in FD medium supplemented with 15% calf serum
13
and OVCAR-3 cells were cultured in RPMI medium supplemented with 20% fetal calf
serum + 1 µg/ml insulin. Culture media were changed every 2-3 days. When the cells
reached near-confluence, they were either utilized for experimentation or passaged (1 out
of 10 ml of trypsinized cells into a new flask).
Determination of Cell Growth Rates
Flasks of cells were trypsinized and aliquots of 25,000-35,000 cells were plated in
quadruplicate wells of 4, 24-well tissue culture plates containing 1.0 ml of appropriate
medium per well. At 1, 2, 3, and 4 days post inoculation, cells in one plate (i.e. 4 wells)
were trypsinized, diluted with appropriate medium to stop the reaction, and then placed
into counting vials. Cell counts were determined using a Coulter Counter and the mean
+/- SEM of the 4 replicates on each day was determined. Data were fitted to a linear
equation and the doubling time for each cell type was determined.
GCNF, Oct-4, GDF-9, and FSH Receptor Oligonucleotide Primer Formulation
GCNF primers were originally generated for use in the hamster for other studies
in our laboratory. Mouse and human mRNA sequences were compared using the NCBI
Website (www.ncbi.nih.gov) and regions were selected that exhibited identity between
the two species. Accordingly, the prepared GCNF primers worked with human samples
and were as follows: sense primer, 5’ GAA CAA GCA ACC TGT CTC AT 3’, and
antisense primer, 5’ ATG GTT GCT CCA GTG ATT G 3’. Following the initial findings
of GCNF expression in ovarian cancer cells, a second human-specific primer set was
generated for eventual analysis of GCNF expression via real-time PCR: sense, 5’ GCT
14
ATG CCT TGG TCC TTG CTG 3’ and antisense, 5’ GTG TTC AAC GGT AGT AAT
GCG 3’. Primers utilized for Oct-4 mRNA analysis were formulated from human
sequences published in GenBank: sense primer, 5’ ATC AAA GCT CTG CAG AAA
GAA CT 3’; antisense primer, 5’ CCT CTC ACT TGG TTC TCG ATA CT 3’. Two
GDF-9 primer pairs were utilized. One pair was designed for use in the hamster (GDF-9
184 and GDF-9 515) and derived from mouse and rat sequences, while the other was
human specific. These primers consisted of: GDF-9 184 sense, 5’ TGT AGA TGG GAC
TGACAG GTC 3’; GDF-9 495; antisense, 5’ TCA GAG TGT ATA GCA AGA CCG 3’;
huGDF-9 up sense, 5’ GCT AAT CCT GCT GTC TTC CC 3’; and huGDF-9 dwn
antisense, 5’ ACT CCT CGT TGC TGA CCT TT 3’. As an ovarian control marker,
primers were designed to identify the mRNA for the follicle-stimulating hormone
receptor (FSH-Rc). These primers consisted of: huFSHR up, 5’ CCC TGC TCC TGG
TCT CTT TGC 3’, and huFSHR dwn, 5’ CAC ATC TGC CTC TAT CAC CTC C 3’.
Primer pairs were selected such that at least one intron lay between the primer sequences
on the gene. This allowed utilization of total RNA for RT/PCR without concern for any
contaminating genomic DNA. Candidate primer pairs were subjected to analysis using
Netprimer (www.premierbiosoft.com/netprimer) to detect inter- and intra-chain primer
dimer formation and intra-chain stem loop formation. Primer specificity was validated
using BLAST analysis. Primers were synthesized by Sigma Genosys and were diluted to
a concentration of 100pmole/µl, aliquoted into small amounts, and stored at –80C.
Primers were diluted to 10pmol/µl as working stocks.
15
RNA Isolation and RT/PCR
The culture media from 75 cm2 flasks of cells were removed and the cells were
rinsed one time with 10 ml phosphate-buffered saline and the flasks were placed on ice.
Three milliliters of Ultraspec Reagent (Biotecx Laboratories, Inc., Houston, TX) were
added to the monolayers to dissolve the cells over a 5-minute period. The lysate was
aspirated several times with a pipette, then split among three, 1.5 ml DEPC-treated
microfuge tubes and the tubes were placed on ice for 5 another minutes to continue the
digestion process. Following this, 0.2 ml of chloroform was added to each tube and the
tubes were shaken vigorously for 15 seconds. The tubes were returned to the ice for
another 5 minutes. The tubes were then centrifuged for 15 minutes @ 12,000 RPM at 4C
using a microfuge. The clear aqueous upper fraction was carefully removed and place
into a fresh microfuge tube. An equal volume of ice-cold isopropanol was then added and
the tubes inverted several times to precipitate total RNA. The tubes were kept at –20C
overnight whereupon the precipitated RNA was concentrated by centrifugation at 12,000
RPM for 30 minutes (4C). The supernatant was removed and the RNA pellet rinsed twice
with 1.0 ml of 75% ethanol in DEPC-treated water. RNA was centrifuged at 7,000 RPM
following each wash. The total RNA pellet was dried by centrifugation for 10 minutes
using a Savant Vacuum Dryer. The RNA pellet was suspended in 25 µl of nuclease-free
water over a 2-hour period at 4C. Following this, the total RNA content was determined
via spectroscopy at 260/280 using an Eppendorf Biophotometer. RNA samples were
stored at –80C until utilized.
Five µg aliquots of total RNA were reverse transcribed using oligo dT and
random hexamer primers and a Reverse Transcription System (Promega Corp., Madison,
16
WI). Reaction samples were incubated at room temperature, 42C, and 94C for 10, 45, and
3 minutes, respectively. Varying amounts of RT product were subjected to PCR using a
Promega PCR Core System I kit and a Thermolyne Amplitron II thermalcycler.
Following an initial 2 minute melt at 94C, samples were subjected to 35 cycles of
primer specific annealing followed by a 72C extension step for 2 minutes. At the end of
the run, the samples were subjected to a 12-minute final extension at 72C after which
they were held at 4C until retrieved. RT/PCR samples were subjected to electrophoretic
separation using a 2% agarose gel containing ethidium bromide. PCR products were
visualized under UV light and photographed using a Kodak EDAS photodocumentation
system.
DNA Sequencing
PCR products were extracted from agarose gels and purified using a QIAEX II
Gel Extraction Kit (Qiagen, Inc., Valencia, CA). DNA content was determined using an
Eppendorf Biophotometer. Approximately 100 ng of purified PCR (25 ng/µl) product
along with relevant primer pairs diluted to 2 pmol/µl were forwarded to the Kansas
University Medical Center Biotechnology Facility in Kansas City, KS, for sequencing.
Immunoblot Detection of GCNF Protein
Whole cell lysates were made by the addition of 1 ml of RIPA buffer (1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, in phosphatebuffered saline, pH 7.4) containing protease inhibitors (Sigma P-8340, 10 µl/ml RIPA
buffer) to sub-confluent 75 cm2 flasks. The cells were detached by scraping with a rubber
17
policeman and the lysates were transferred to bullet tubes then aspirated vigorously to
dissolve cell contents. Lysates were incubated on ice for 45 minutes then centrifuged at
10,000 RPM for 10 minutes at 4C. The cleared lysates were aspirated and stored at –20C.
Lysate protein content was determined using a BRC Protein Assay kit from BioRad
modified for microsamples. Lysates were diluted 4-to-5 with 5x Laemmli sample buffer
(16) without sulfydryl reducing agents and heated @ 94C for three minutes. Volumes of
lysates were added to a pre-cast, 10% acrylamide/Tris-glycine, Novex mini-gel
(Invitrogen, Carlsbad, CA), such that the protein content added was equivalent among the
samples. In addition, prestained molecular weight markers (Invitrogen) and 2nd antibodysensitive molecular weight markers (Santa Cruz Biotechnology, Santa Cruz, CA) were
also
run.
Following
electrophoretic
separation,
proteins
were
transferred
electrophoretically to PVDF hydrophobic membranes. Membranes were blocked for 6090 minutes with Blotto A. Following this, the membranes were exposed to primary
antibody for 60-90 minutes at room temperature. A polyclonal rabbit anti-mouse GCNF
kindly provided by Dr. Austin Cooney (Dept. of Molecular and Cellular Biology, Baylor
College of Medicine), was utilized as primary antibody diluted 1:500 with Blotto A.
Following this, the membranes were washed with TBS/Tween 20 and then incubated
with goat anti-rabbit Ig linked to horseradish peroxidase for 1 hour. Following washing
with TBS/Tween 20, the membranes were treated with Luminol reagent (Santa Cruz
Biotechnology) for 1 minute. Chemiluminescent band development was followed using a
BioRad Versadoc digital photoimaging system.
18
Impact of Retinoic Acid upon Ovarian Cancer Cell GCNF mRNA Expression
TOV-112 and ES-2 cells (~200,000/well) were each cultured in 4 wells of a 6well culture dish in 4.0 ml of F-12: DMEM (1:1) +20% fetal calf serum + 1 µg/ml insulin
and McCoy’s 5A medium + 10% fetal calf serum, respectively, for 24 hours. Following
this, the medium was replaced with fresh respective medium alone, medium containing
DMSO at a final concentration of 0.1%, or medium containing 10-6 or 10-5M all trans
retinoic acid (dissolved in DMSO which achieved a final concentration of in 0.1% in the
medium). After 2 days of treatment, the cells were subjected to total RNA isolation. The
total RNA concentration of the samples was 2.53-to-3.05 µg/µl and the total RNA content
among the samples was 63.3-to-76.3µg. Total RNA samples (7.5µg) were subjected to
reverse transcription using oligo dT and random hexamer primers. Samples of RT
product were subjected to PCR as described above using GCNF oligonucleotide primers
and an annealing temperature of 55C. Equal aliquots of PCR product were subjected to
agarose gel electrophoresis with ethidium bromide. PCR product bands were digitally
analyzed using a Kodak EDAS photodocumentation system and the bands expressed as
mean fluorescence intensity.
SiRNA Transfection
In a six well tissue culture plate, ES-2 and TOV-112 cells were seeded with 2 ml
antibiotic-free normal growth medium supplemented with FCS per well. The cells were
incubated at 37° C in a CO2 incubator until the cells are 60 - 80% confluent. This will
usually take 18-24 hours. Healthy and subconfluent cells are required for successful
transfection experiments. The cell viability was ensured one day prior to transfection.
19
Solution A was prepared for each transfection, by diluting 8 µl of siRNA duplex
(i.e., 0.25-1 µg or 20-80 pmols siRNA) into 100 µl siRNA Transfection Medium.
Solution B was prepared by diluting 8 µl of siRNA Transfection Reagent into 100
µl siRNA Transfection Medium: Peak activity should be at about 6 µl siRNA
Transfection Reagent. Serum and antibiotics shouldn’t be added to the siRNA
Transfection Medium. The siRNA duplex solution (Solution A) was then directly added
to the dilute Transfection Reagent (Solution B) using a pipette. The solution was then
mixed gently by pipetting the solution up and down and the mixture was incubated for
15–45 minutes at room temperature.
Then the cells were washed once with 2 ml of siRNA Transfection Medium: and
immediately aspirated.
For each transfection, 0.8 ml siRNA Transfection Medium should be added to
each tube containing the siRNA: Transfection reagent mixture. Then it was mixed gently
and the mixture was overlaid onto the washed cells. The cells were then incubated for 9
hours and thirty minutes at 37° C in a CO2 incubator.
Following this, 1 ml of normal growth medium was added containing 2 times the
normal serum and antibiotics concentration (2x normal growth medium) without
removing the transfection mixture. If toxicity is a problem, the transfection mixture was
removed and replaced with 1x normal growth medium.
The cells were then incubated for an additional 18–24 hours after which the
medium was aspirated and replaced with fresh 1x normal growth medium. After all these
steps, the cells
were assayed using the appropriate protocol 24–72 hours after the
addition of fresh medium in the step above.
20
The control siRNA, GCNF siRNA and all its accessory reagents were synthesized
for us by Santa Cruz Biotechnology.
A Conserved Domain Database and Search Service, v2.09
Proteins generally contain several modules or domains, each with a distinct
evolutionary origin and function. NCBI's Conserved Domain Database is a collection of
multiple sequence alignments for domains and full-length proteins. The CD-Search
service is used to identify the conserved domains present in a protein query sequence.
Computational biologists characterize conserved domains based on recurring
sequence patterns or motifs. The un-curated section of CDD contains domains imported
from Pfam, SMART and COGs. The source databases also provide brief explanations and
links to citations. Because conserved domains correspond to compact structural units,
CDs are linked to 3D structures. Thus the NCBI-curated section of CDD attempts to
group ancient domains related by common descent into family hierarchies.
To identify conserved domains in a protein sequence, the CD-Search service
makes use of the reverse position-specific BLAST algorithm. The query sequence is
compared to a position-specific score matrix prepared from the underlying conserved
domain alignment. Hits are displayed as pairwise alignments of the query sequence with
representative domain sequences, or as multiple alignments. Thus the CD-Search is run
by default in parallel with protein BLAST searches.
Cn3D 4.1
Cn3D is basically a visualization tool for biomolecular structures, sequences, and
sequence alignments. Cn3D displays structure-structure alignments along with their
21
structure-based sequence alignments, to emphasize the regions of a group of related
proteins which are most conserved in structure and sequence. Cn3D is typically run from
a WWW browser as a helper application for NCBI's Entrez system, but it can also be
used as a stand alone application. With version 4, Cn3D is now a complete multiple
alignment editor as well, and includes algorithms for aligning sequences to other
sequences and to structures. Cn3D is used as the primary alignment curation tool for the
CDD project.
Statistical Analysis:
Data were analyzed by a one-way ANOVA, where appropriate. And the comparisons
among groups were tested using a Tukey-Kramer Multiple Comparison Test. Statistical
analyses were generated using Instat software.
22
RESULTS
GCNF mRNA Expression in the Normal Hamster Ovary
Our initial studies were performed to investigate the expression of GCNF in the
hamster ovary to monitor developmental changes. As the hamster genome is not
sequenced, the oligonucleotide primers used were designed from consensus sequences
taken from mouse and human GCNF mRNAs. Since GCNF has been reported to be
largely relegated to the germ line in adults we tested the utility of the oligonucleotide
primers in amplifying GCNF from the hamster ovary. The results contained in Figure1
indicate clearly that hamster ovaries from 11-to-298 days old express GCNF mRNA,
which was determined by RT/PCR. The bands generated were consistent with the
predicted PCR product of 351 bp generated using the designed primers for GCNF.
To validate the PCR product, the hamster ovarian mRNA bands were extracted
from the gels and sequenced. The results contained in Figure 1 illustrate the degree of
sequence homology between the hamster ovarian PCR product and the mouse GCNF
mRNA sequence contained in Genbank (gi623257). The sequences yielded 94%
homology (333/351 bp) strongly suggesting that the PCR products generated by the
consensus primers is indeed GCNF.
Characteristics of Ovarian Cancer Cell Lines Used
Several human ovarian cancer cell lines were tested for GCNF expression. The
cell lines used were two ovarian epithelial adenocarcinoma lines (TOV-112D, ES-2), one
epithelial clear-cell carcinoma (OVCAR-3) and one germ cell teratocarcinoma line
23
(PA-1). The cell lines exhibited distinct morphologies, when maintained in monolayer
culture (Figure 2). ES-2 and PA-1 cells displayed a fibroblastic, spindle shaped
morphology and grew largely as single cells. TOV-112 cells were less fibroblastic but
also grew as single cells. In marked contrast, the OVCAR-3 cells, the slowest growing by
far were large, polyhedral shaped cells which grew as colonies. These 4 cell lines exhibit
markedly different growth rates (Figure 2). OVCAR-3 cells grew very slowly exhibiting
a doubling time of 5-6 days. In contrast, TOV-112D and CRL-1572 cells grew much
more rapidly exhibiting doubling times of ~ 0.9 days. Lastly, ES-2 cells were the fastest
growing ones, exhibiting a doubling time of ~0.5 days.
Expression of GCNF mRNA in Ovarian Cancer Cell Lines
To demonstrate possible GCNF expression in the above cell lines, total cell RNA
was isolated, reverse transcribed, and subjected to PCR using the GCNF primers. The
results showed in Figure 3 indicate that all four cell lines express a PCR product
consistent with the predicted 351 bp GCNF product. Although they had differences in
their neoplastic origin and possessed markedly different growth rates in vitro, all the
ovarian cancer cell lines used, expressed GCNF mRNA. To validate our results, the
TOV-112 PCR product was sequenced
and compared to the human GCNF mRNA
sequence contained in Genbank which exhibited 100% homology (351/351 bp, Figure 4).
Thus, the ovarian cancer cell lines appear to express intact and authentic GCNF, at least
in the DNA binding domain.
24
Expression of GCNF protein by ovarian cancer cell Lines
Western blot analysis was performed on the whole cell lysates prepared from ES2, TOV-112, and PA-1 cells using a rabbit anti-mouse GCNF antibody directed against
the amino terminus. Non-denaturing, non-reducing conditions were used in an attempt to
minimize alterations in GCNF structure including multimeric forms of the receptor. All
three cell lines expressed a strong band which corresponded to a molecular weight of
~135 kD (Figure 3, large arrow) as determined from a plot of the Rf values for the
molecular weight (MW) standards as a function of the log of their molecular weights.
This apparent molecular weight is consistent with GCNF homodimerization of the
reported 58-61 kD monomer in the mouse (Lane 3, 17). Other bands were also observed
particularly in the PA-1 cells corresponding to molecular weights of ~56, 82, and 107 kD.
A large band was observed when the molecular weight curve was extrapolated, which
correlates with an approximate molecular weight of 360-400 kD (Figure 3, small arrow).
Immunoblotting using of an equal amount of non-immune rabbit Ig to probe the
membranes did not reveal any of the bands described above suggesting the bands
detected by the anti-GCNF antibody represent actual forms of the nuclear receptor.
Expression of GDF-9 and Oct-4 by ovarian cancer cell lines
A key cellular role for GCNF is the repression of other genes including the stem
cell pluripotency factor, Oct-4, and the growth regulatory factor GDF-9. So it would
seem logical that, if the GCNF expressed in the ovarian cancer cells is functionally
normal, then neither Oct-4 nor GDF-9 would be expressed. Total RNA from ovarian
cancer cells was prepared and subjected to RT/PCR using Oct-4- and GDF-9-specific
25
primers described in the Methods. The results contained in Figure 5 indicate that both
Oct-4 and GDF-9 are expressed at least at the mRNA level in these cells. Thus, not only
is GCNF inappropriately expressed in these cells, its regulatory activity appears to be
altered. Selective cancer cell mRNA expression utilizing human-specific primers is
illustrated in Figure 5 using ES-2 and TOV-112D cells where transcripts for GCNF,
GDF-9, and Oct-4 are observed but not follicle-stimulating hormone (FSH) receptor. This
suggests that there exists selective expression of genes in these cells.
Impact of retinoic acid upon GCNF expression
Retinoic acid (RA), a derivative of vitamin A has been found to stimulate GCNF
expression variably and transiently in various cells [82]. Based on this fact, we were
inquisitive of the impact retinoic acid would have on GCNF expression in the cancer cell
lines. Accordingly, TOV-112, ES-2, and OVCAR-3 cells were each cultured in their
respective media for 24 hours. Following this, the media were replaced with fresh
respective media alone, media containing DMSO at a final concentration of 0.1%, or
medium containing 10-6 or 10-5M all trans-retinoic acid (dissolved in DMSO which
achieved a final concentration of in 0.1% in the medium). After 48 hours, total cellular
RNA was isolated and subjected to RT/PCR for GCNF. The data illustrated in Figure 6
indicate that retinoic acid stimulated the expression of GCNF mRNA compared to the
DMSO vehicle control. In all the three groups the mean increase in GCNF mRNA
relative to the DMSO control was 70.7+/- 30.8% at 10-5M RA. Retinoic acid has been
reported to suppress Oct-4 expression in cells leading to loss of pluripotency and
initiation of cell differentiation. GCNF has further been suggested as a possible mediator
26
of this effect. Since RA stimulated GCNF expression in ES-2 and TOV-112D cells and
GCNF normally suppresses Oct-4, it would seem logical that RA should decrease Oct-4
expression. However, there was little effect upon Oct-4 expression despite the positive
effect of RA on GCNF expression (Figure 6). Thus, it would appear that GCNF
expressed in these cancer cells has lost its regulatory activity with regard to Oct-4 as well
as GDF-9 (Figure 5).
GCNF Gene knock down (silencing) to determine the effects of GCNF on Growth
Armed with the siRNA probe (Synthesized by Santa Cruz Biotechnology) and
using procedures outlined by Santa Cruz Biotechnology, ES-2 and TOV-112 cells were
transfected with GCNF siRNA as well as with the control siRNA. Seventy two hours
post-transfection, cell count levels were determined using the Coulter Counter and the
cellular RNA content was analyzed by RT/PCR respectively. Relative to untreated and
control siRNA-treatments, GCNF siRNA-treatment decreased the monolayer cell content
by 45.6% and 36.2% for ES-2 and TOV-112 cells, respectively (p<0.001), (Tables 1-3).
Consistent with the observed decline in cell proliferation, GCNF siRNA treatment
decreased the cellular content of GCNF mRNA by 37.6% in TOV-112 cells (p<0.05),
(figure 7).
27
Experiment to determine the effects of Growth suppression on GCNF expression
The non-transformed mink lung epithelial cell line (MLEC) expresses GCNF.
Importantly, MLEC are both contact inhibited and exquisitely sensitive to TGF-β1, which
markedly inhibits proliferation. This is in contrast to PA-1 cells which are not inhibited
by TGF-beta. Both MLEC and PA-1 were cultured in the presence and absence of TGFβ1 (1 and 5ng) for 48hrs. The cell counts were then determined using Coulter Counter
which showed a significant decrease in the TGF-beta treated cells (53.7% and 69.9%
decrease at 1 and 5ng/ml respectively) compared to the control, but only in the MLEC
cells (p<0.001) (Table 4) and not in PA-1 cells (Table 6). To investigate whether this
inhibition in growth and proliferation had any effect on the GCNF mRNA expression,
RNA was isolated from the cells at the same time point as above and RT/PCR was
performed. The total RNA levels for MLEC exhibited a 77% and 88% decrease with 1ng
and 5ng TGF-β relative to control (Table 5). Equal volumes of RNA were used to carry
out RT and then PCR was performed using GCNF specific primers. The results in Figure
8 show that there is a dose dependent attenuation in the GCNF mRNA expression which
corresponds to the decrease in the proliferation upon TGF-beta treatment. Although we
cannot rule out whether this attenuation in the GCNF expression is through direct
activation of the TGF-beta pathway, we hypothesize that GCNF is related to growth
which is supported by the fact that GCNF is expressed in rapidly proliferating tissues and
cells.
28
DISCUSSION
Based on phylogenetic studies [83], the nuclear receptor superfamily is
subdivided into 6 subfamilies and 26 groups of receptors. Interestingly, the subfamily VI
is defined by GCNF as the only member identified so far. The orphan receptor germ cell
nuclear factor (GCNF) is a member of the superfamily of nuclear receptors which is
primarily expressed during embryogenesis. GCNF expression is restricted to the
developing nervous system, whereas in the adult, the receptor is also expressed during
specific stages in maturing germ cells of the ovary and testis. Modifications and missexpressions of nuclear receptors including steroid receptors, thyroid receptors, retinoid
receptors, and others, have been linked to many neoplasias [84].
The discovery of GCNF expression in ovarian cancer cells was very interesting
based upon the restricted expression pattern for GCNF within the germ line in adult
mammals and we were inquisitive whether this transcription factor might be expressed in
germ cell tumors. The finding that GCNF is expressed in ovarian cancer cells of both
epithelial and germ cell origin suggest that it may function as a general regulatory factor
perhaps facilitating cell proliferation. Conventionally, however, GCNF is not associated
with having a direct role in cell proliferation. Rather, its role is associated with embryonic
developmental changes and gametogenesis. Most importantly, GCNF is essential for
embryonic survival in that GCNF null mice only survive through midgestation likely due
to cardiac failure [85]. GCNF has also been reported to suppress the expression of GDF-9
and BMP-15, two members of the TGF-ß superfamily that function during ovarian
follicle development [86]. Also significantly lower amounts of the 7.5 kb mRNA are
29
detected in other rapidly proliferating tissues such as ovary, liver, kidney, lung, and brain
[87, 88]. This would suggest a proliferative role for GCNF.
Since neither potential ligand, heterodimerization partners, nor cofactors for
GCNF have been identified, little is known about the mechanisms by which the receptor
controls transcriptional processes and how it contributes to the neoplastic state. One thing
we know best about GCNF is that retinoic acid (RA) is a key regulatory factor. RAmediated induction of GCNF is thought to be a primary and a critical factor for primary
neurogenesis (89). RA requires GCNF to induce differentiation of embryonic stem cells
and P19 embryonal carcinoma cells [90]. Our results also suggest that despite a basal
level of GCNF expression by ovarian cancer cells, RA can be induced above its basal
expression. While RA can certainly upregulate GCNF expression, it is unlikely that RA is
a key factor promoting GCNF expression in the ovarian cancer cells. Owing to the fact
that GCNF expression is indeed regulated developmentally, it is not unreasonable that
cancer cells express it as they express other embryonic proteins.
Of paramount importance in addition to the discovery of GCNF expression in
ovarian cancer cell lines, is its apparent inability to function normally in terms of the
repression of target genes such as Oct-4 and GDF-9. Oct-4 has been shown to be one of
the master regulatory genes maintaining the pluripotency of stem cells [91]. Repression
of Oct-4 expression is consistent with loss of pluripotency and the initiation of cell
differentiation. Retinoic acid-mediated differentiation of P19 embryonal carcinoma cells
was characterized by the disappearance of Oct-4 [92]. Oct-4 loss-of-function mutation
results in the differentiation of embryonic epiblast cells into trophectoderm [93].
30
Oct-4 expression in cancer cells is not very surprising due to the increasing
recognition of the existence of cancer stem cells that could perpetuate cancer cell
“differentiation” characterized by unregulated growth [94, 95, 96]. In embryonic stem
cells, the level of Oct-4 expressed appears to determine the fate of cells in terms of selfrenewal, differentiation, or dedifferentiation [97]. Thus, Oct-4 is largely related to cancer
propagation and may underlie the process of cancer recurrence. With regard to Oct-4,
however, one demonstrated regulator is GCNF. Overexpression of GCNF cells represses
Oct-4 expression promoting cell differentiation in P19 embryonal carcinoma [98].
Developmentally, GCNF and Oct-4 expression are reciprocally related (12). GCNF is
required for pluripotency gene repression associated with retinoic acid-mediated
differentiation of embryonic stem cells [99]. Thus, the finding that GCNF and Oct-4 as
well as GDF-9 are expressed in these cancer cells strongly suggests an altered/impaired
regulatory activity.
Since GCNF is a transcriptional repressor, a logical location for mutation would
involve the DNA binding domain. Any mutation affecting the ability of the repressor
protein to bind target DNA would facilitate gene expression. However, the integrity of at
least the majority of the DNA binding domain of cancer cell GCNF appears to be normal
since the sequence homology of the PCR product was identical to the published sequence
(Figure 4). The functional gene sequence spanned by the mouse/human GCNF primers
utilized in this study (Figure 4) included 90% of exon 4 encoding the DNA binding
domain [100,101] and all of exon 5 which, is involved in homodimeric interactions of the
receptor. Thus, it appears that the DNA binding domain is relatively normal in the cancer
cell GCNF. It is also important to note that we identified, specific protein bands
31
consistent with homodimerization and oligomerization of the GCNF monomer by
western blot analysis (Figure 3C). This is in contrast to P19 embryonal carcinoma cells in
which neither a homodimer nor a TRIF complex (GCNF hexamer oligomerization) can
be found without associated DNA [102]. The lesion involving GCNF may reflect the
formation of multimers prior to association with its DNA binding site and this altered
GCNF might not be able to silence genes properly. Recently, it has been demonstrated
that GCNF induces long-term silencing of Oct-4 via recruitment of DNA
methyltransferase which induces methylation in the Oct-4 promoter. This explains the
importance of GCNF in regulating pluripotency and its probable role in cancer when it is
expressed in an altered form [103].
In addition to different Oct-4 and GDF-9 expression patterns in these ovarian
cancer cell lines, we have also shown that GCNF is directly or indirectly involved with
growth and proliferation. We used siRNA technology to knock down GCNF mRNA
which was correlated to a decrease in proliferation in two of the ovarian cancer cell lines
tested (ES-2 and TOV-112) by approximately 40%. We have also reported that there is
attenuation in GCNF mRNA expression when the cell proliferation was suppressed in the
Mink Lung Epithelial cells by adding various amounts of TGF-beta 1 (1 and 5ng) to the
culture. This attenuation in the GCNF mRNA expression is probably a growth related
effect and not via the TGF-beta signaling mechanism as GCNF is expressed at its fullest
in the rapidly growing cells and tissues. However these experiments suggest a strong
relationship between cell proliferation, differentiation and GCNF.
There are also studies relating inhibition of cell proliferation by TGF-beta, to
binding of retinoic acid related factors to its promoter regions which can cause inhibition.
32
It is known that transforming growth factor beta 1 (TGF beta 1) is the prototype of a large
family of polypeptides involved in growth control, extracellular matrix production, and
development. TGF beta 1 expression is largely governed by three AP-1 binding sites
located in two different promoters of this gene. AP1 regulates the expression of several
genes involved in oncogenic transformation and cellular proliferation such as those
coding for metalloproteases, VEGF, and transforming growth factor ß (TGF-ß), and AP1
transactivation is required for tumor promotion in vivo (104).
Recent studies have examined the ability of retinoid receptors to inhibit the activity
of the two promoters by cotransfection assays in the hepatocellular carcinoma cell line
HepG2. When the TGF beta 1 promoter activity is induced by 12-O-tetradecanoyl
phorbol13-acetate (an activator of AP-1-controlled gene transcription), this activity can
be strongly repressed by retinoic acid receptor-alpha (RAR alpha), RAR beta, or retinoid
X receptor-alpha (RXR alpha) as well as other members of the nuclear receptor family.
On further examining the anti-AP-1 activity of RXR alpha, it was observed that three
different AP-1-controlled promoters (TGF beta 1, collagenase, and cFos) can be
inhibited. Using gel shift assays, it was demonstrated that RXR alpha inhibits Jun and Fos
DNA binding and that 9-cis RA enhances this inhibition, suggesting that a mechanism
involving direct protein-protein interaction between RXR and AP-1 components mediates
the inhibitory effect observed in vivo. Transfection analyses with RXR alpha point
mutations revealed that residues L422, C432, and, to a lesser extent, residues L418 and
L430, are involved in ligand-induced anti-AP1 activity of RXR alpha in vivo. Thus, both
types of retinoid receptors can inhibit AP-1-activated promoters, including the TGF beta
1 gene promoter, via a mechanism that involves protein-protein interaction which opens
33
up novel assumptions that the TGF-beta inhibition of the MLEC cells and many other
cells and cell lines observed by various other labs might be due to the binding of the
normal GCNF (which shares high degree of homology, at least structurally with RXR) to
the promoters in TGF-beta. The fact that many cancer cell lines are insensitive to TGFbeta treatment can very well explain the possibility that a mutated and a functionally
abnormal GCNF is bound to the TGF-beta and various other promoters which have a
regulatory effect on cell growth and proliferation. Though GCNF has not been found so
far to have heterodimeric partners in normal cells, there is always a possibility of it
forming heterodimers in its mutated version found in the cancer cell lines which can also
explain our immunoblot results consistent with some high molecular weight bands along
with monomeric forms [105].
Studies with regard GCNF DNA and Ligand binding domains were also
performed in our lab by using CDD (Conserved Domain Database) [41] and Cn3D (a
visualization tool for 3 dimensional structure) [42]. The DNA binding domain of GCNF
shares a 3D homology with 1HLZ which is homo sapiens chain A crystal structure of
orphan nuclear receptor Rev-Erb (Alpha), named because it is encoded on the opposite
strand of the T3R
(Figure 9). It is also a strong repressor of N-Myc expression
suggesting an important role in the control of cell proliferation and organ physiology
[43].
The ligand binding domain shares a high degree of structural homology (3D) with
1HG4 chain A, ultraspiracle ligand binding domain from Drosophila (Figure 10). As USP
has RXR as its mammalian homologue, GCNF (the ligand binding domain) shares a high
degree of homology with RXR than with any other nuclear receptor. The structure of the
34
ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive
conformation.
Ultraspiracle (USP) is the invertebrate homologue of the mammalian retinoid X
receptor (RXR). RXR plays a uniquely important role in differentiation, development,
and homeostasis through its ability to serve as a heterodimeric partner to many other
nuclear receptors. RXR is able to influence the activity of its partner receptors through
the action of the ligand 9-cis retinoic acid. In contrast to RXR, USP has no known highaffinity ligand and is thought to be a silent component in the heterodimeric complex with
partner receptors such as the ecdysone receptor. The structure shows that a conserved
sequence motif found in dipteran and lepidopteran USPs, but not in mammalian RXRs,
serves to lock USP in an inactive conformation. It also shows that USP has a large
hydrophobic cavity, implying that there is almost certainly a natural ligand for USP. This
cavity is larger than that seen previously for most other nuclear receptors. Intriguingly,
this cavity has partial occupancy by a bound lipid, which is likely to resemble the natural
ligand for USP [44].
Previous studies have demonstrated that posttranslational modifications of RARs
and RXRs are critical to the biological activity of the receptors. Modifications such as
phosphorylation [106, 107] and mutations that mimic the effect of ligands [108] can also
induce conformational changes within the LBD which generate constitutive active
receptors. The N-terminal domain (NTD) is also not well conserved within the nuclear
receptor superfamily. In some cases the NTD harbors a cell type and/or promoter-specific
transcriptional activation function (AF-1) [109,110,111]. The activity of the AF-1 can be
regulated by secondary modifications such as phosphorylation [109,110,111]
35
There are many phosphorylation sites (5 Protein kinase C phosphorylation sites, 10
Casein kinase II phosphorylation sites and 1 Tyrosine kinase phosphorylation site)
predicted by the Prosite scan in both DNA and the ligand binding domains of GCNF. The
phosphorylations in these domains might account and explain the huge multimeric
associations in our immunoblot which can be either just the GCNF monomers linked
together or bound with co-repressors or even heterodimers associated with promiscuous
nuclear receptors like RXR. And any alteration in these sites can potentially inhibit the
normal regulatory action of GCNF and knock out its whole ability in binding with AP-1
or AP-1 like sites which are very important in maintaining cell proliferation and growth.
Since it is possible that GCNF can be misexpressed and can mismanage the so
called “pluripotency genes”, it is worth speculating its role in oncogenesis. In summary,
the ligand for GCNF and how it contributes to the neoplastic state are largely unknown.
However, this is the first step toward verifying its difference in functional
characterization and its uniqueness as a nuclear receptor both structurally and
functionally which opens up new doors for further research.
36
REFERENCES
37
LIST OF REFERENCES
1.
Revelli A, Massobrio M, Tesarik J (1998) Nongenomic actions of steroid
hormones in reproductive tissues. Endocr Rev 19:3–17
2. Chambon P (1996) A decade of molecular biology of retinoic acid receptors.
Whelan WJ (ed) The Retinoid Revolution. FASEB J 10:940–954.
3. Tora L, Gronemeyer H, Turcotte B, Gaub MP, Chambon P (1988) The N-terminal
region of the chicken progesterone receptor specifies target gene
activation. Nature 333:185–188
4. Berry M, Metzger D, Chambon P (1990) Role of the two activating domains of the
oestrogen receptor in the cell-type and promoter-context dependent agonistic activity
of the anti-oestrogen 4-hydroxytamoxifen.EMBO J 9:2811–2818
5. Glass CK (1994) Differential recognition of target genes by nuclear receptors
monomers, dimers, and heterodimers. Endocr Rev 15:391–407
6. Mangelsdorf DJ, Evans RM (1995) The RXR heterodimers and orphan receptors.
Cell 83:841–850
7. Yen PM, Sugarawa A, Chin WW (1992) Triiodothyronine (T3) differentially
affects T3-receptor/retinoic acid receptor and T3-receptor/retinoid X receptor
heterodimer binding to DNA. J Biol Chem 267:23248–23252
8. Umesono K, Evans RM (1989) Determinants of target gene specificity for
steroid/thyroid hormone receptors. Cell 57:1139–1146
9. Zechel C, Shen X-Q, Chambon P, Gronemeyer H (1994) Dimerization interfaces
formed between the DNA binding domains determine the cooperative binding of
RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.
EMBO J 13:1414–1424
10. Sap J, Muñoz A, Schmitt J, Stunnenberg H, Vennström B (1989) Repression of
transcription mediated at a thyroid hormone response element by the v-erb-A
oncogene product. Nature 340:242–244
11. Beato M, Sánchez-Pacheco A (1996) Interaction of steroid hormone receptors with
the transcription initiation complex. Endocr Rev 17:587–609
12. Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE, Schreiber SL,
Evans RM (1997) Nuclear receptor repression mediated by a complex containing
SMRT, mSin3a, and histone deacetylase. Cell 89:373–380
38
LIST OF REFERENCES (continued)
13. Bannister AJ, Kouzarides T (1996) The CBP co-activator is a histone
acetyltransferase. Nature 384:641–643
14. Wolffe AP (1997) Transcriptional control—sinful repression. Nature 387:16–17.
15. Wong J, Shi Y-B, Wolffe AP (1997) Determinants of chromatin disruption and
transcriptional regulation instigated by the thyroid hormone receptor:hormone
regulated chromatin disruption is not sufficient for transcriptional activation.
EMBO J 16:3158–3171
16. Jensen EV, Jacobson HI, Flesher JW, Saha NN, Gupta GN, Smith S, Colucci V,
Shiplacoff D, Neuman HG, Desombre ER, Jungblut PW (1996) Estrogen
receptors in target tissues. In: Pincus G, Nakao T, Tait JF (eds) Steroid
Dynamics. Academic Press, New York, pp 133–156
17. Sporn MB, Roberts AB (1983) Roles of retinoids in differentiation and
carcinogenesis. Cancer Res 43:3034–3040
18. Ashburner M, Chihara C, Meltzer P, Richards G (1974) Temporal control of
puffing activity in polytene chromosomes. Cold Spring Harbor Symp Quant
Biol 38:655–662
19. Thompson CC, Weinberger C, Lebo R, Evans RM (1987) Identification of a novel
thyroid hormone receptor expressed in the mammalian central nervous system.
Science 237:1610–1614
20. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-Å (1996)
Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc
Natl Acad Sci USA 93:5925–5930
21. Mosselman S, Polman J, Dijkema R (1996) ERß: identification and
characterization of a novel human estrogen receptor. FEBS Lett 392:49–53
hormone- EMBO J 16:3158–31
22. Agoulnik IY, Cho Y, Niederberger C, Kieback DG and Cooney AJ (1998) Cloning,
expression analysis and chromosomal localization of the human nuclear receptor
gene GCNF. FEBS Lett, 424, 73–78.
23. Chung AC, Katz D, Pereira FA, Jackson KJ, DeMayo FJ, Cooney AJ and O'Malley
BW (2001) Loss of orphan receptor germ cell nuclear factor function results in
ectopic development of the tail bud and a novel posterior truncation. Mol Cell
Biol, 21, 663– 677.
39
LIST OF REFERENCES (continued)
24. Katz D, Niederberger C, Slaughter GR and Cooney AJ (1997) Characterization of
germ cell-specific expression of the orphan nuclear receptor, germ cell
nuclearfactor. Endocrinology, 138, 4364–4372.
25. Susens U, Aguiluz JB, Evans RM and Borgmeyer U (1997) The germ cell nuclear
factor mGCNF is expressed in the developing nervous system. Dev Neurosci, 19,
410-420.
26. Chen F, Cooney AJ, Wang Y, Law SW, Oalley BW. Cloning of novel orphan
Receptor (GCNF) expressed during germ cell development. Mol Endocrinol 1994; 8:
1434-44.
27. Zhang YL, Akmal KM, Tsuruta JK, Shang Q, Hirose T, Jetten AM, Kim KH and
O'Brien DA (1998) Expression of germ cell nuclear factor (GCNF/RTR) during
spermatogenesis. Mol Reprod Dev, 50, 93–102.
28. Otsuka F, Yao Z, Lee T, Yamamoto S, Erickson GF and Shimasaki S (2000) Bone
morphogenetic protein-15. Identification of target cells and biological functions. J
Biol Chem, 275, 39523–39528.
29. Cooney AJ, Hummelke GC, Herman T, Chen F and Jackson KJ (1998) Germ cell
nuclear factor is a response element-specific repressor of transcription. Biochem
Biophys Res Commun, 245, 94–100.
30. Nuclear Receptors Nomenclature Committee (1999). A unified nomenclature
system for the nuclear receptor superfamily. Cell 97,1 -3.
31. Elvin JA, Clark AT, Wang P, Wolfman NM and Matzuk MM (1999a) Paracrine
Actions of growth differentiation factor-9 in the mammalian ovary.
Mol Endocrinol, 13, 1035–1048.
32. Greschik H, Wurtz JM, Hublitz P, Kohler F, Moras D, Schule R (1999)
Characterization of the DNA-binding and dimerization properties of the nuclear
orphan receptor germ cell nuclear factor. Mol Cell Biol; 19:690 703.
33. Pratt WB, Toft DO (1997) Steroid receptor interactions with heat shock protein
and immunophilin chaperones. Endocr Rev 18:306–36.
34. Baniahmad A, Köhne AC, Renkawitz R (1992) A transferable silencing domain is
present in the thyroid hormone receptor, in the v-erbA oncogene product and in
the retinoic acid receptor. EMBO J 11:1015–1023
35. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L (1996)
Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177.
40
LIST OF REFERENCES (continued)
36. Beato M, Sánchez-Pacheco A (1996) Interaction of steroid hormone receptors with
the transcription initiation complex. Endocr Rev 17:587–609.
37. Glass CK, Rose DW, Rosenfeld MG (1997) Nuclear receptor coactivators.
Curr.Opin.Cell Biol 9:222–232
38. Bannister AJ, Kouzarides T (1996) The CBP co-activator is a Histone
acetyltransferase. Nature 384:641–643
39. Wong J, Shi Y-B, Wolffe AP (1997) Determinants of chromatin disruption and
transcriptional regulation instigated by the thyroid hormone receptor:
40. H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N.
Shindyalov, P.E. Bourne (2000) The Protein Data Bank. Nucleic Acids Research, 28
pp. 235-242.
41. Marchler-Bauer A et al (2005) CDD: a Conserved Domain Database for protein
classification. Nucleic Acids Res. 2005;33 Database Issue:D192-6.
42. Panchenko AR, Madej T (2005) Structural similarity of loops in protein
families: toward the understanding of protein evolution. BMC Evol. Biol 5:10
43. Sierk, M.L., Zhao, Q., Rastinejad, F. (2001) DNA Deformability as a Recognition
Feature in the RevErb Response Element Biochemistry v40 pp.12833-12843.
44. Clayton, G.M., Peak-Chew, S.Y., Evans, R.M., Schwabe, J.W (2001) The structure
of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an
inactive conformation. Proc.Natl.Acad.Sci.USA v98 pp.1549-1554.
45. Lazar MA, Hodin RA, Darling DS, Chin WW (1989) A novel member of the
thyroid/steroid hormone receptor family is encoded by the opposite strand of
the rat c-erbA transcriptional unit. Mol Cell Biol 9:1128
46. Miyajima N, Horiuchi R, Shibuya Y, Fukushige S-i Matsubara K-i Toyoshima
K, Yamamoto T (1989) Two erbA homologs encoding proteins with different T3
binding capacities are transcribed from opposite DNA strands of the same
genetic locus. Cell 57:31–39
47. Forman B, Chen J, Blumberg B, Kliewer SA, Henshaw R, Ong ES, Evans RM
(1994) Cross-talk among ROR 1 and the Rev-erb family of orphan nuclear
receptor. Mol Endocrinol 8:1253–1261
41
LIST OF REFERENCES (continued)
48. Dumas B, Harding HP, Choi H-S, Lehman KA, Chung M, Lazar MA, Moore DD
(1994) A new orphan member of the nuclear hormone receptor superfamily
closely related to Rev-Erb. Mol Endocrinol 8:996–1005
49. Bonnelye E, Vanacker J-M, Desbiens X, Begue A, Stehelin D, Laudet V (1994) Reverbß, a new member of the nuclear receptor superfamily, is expressed in the
nervous system during chicken development. Cell Growth Differ 5:1357–1365
50. Enmark E, Kainu T, Pelto-Huikko M, Gustafsson J-Å (1994) Identification of a
novel member of the nuclear receptor superfamily which is closely related to
rev-erbA. Biochem Biophys Res Commun 204:49–56.
51. Harding HP, Lazar MA (1993) The orphan receptor Rev-ErbA activates
transcription via a novel response element. Mol Cell Biol 13:3113–3121
52. Harding HP, Lazar MA (1995) The monomer-binding orphan receptor Rev-erb
epresses transcription as a dimer on a novel direct repeat
Mol Cell Biol 15:4791–4802
53. Adelmant G, Begue A, Stehelin D, Laudet V (1996) A functional Rev-Erbresponsive element located In the human Rev-Erb- promoter mediates a
repressing activity. Proc Natl Acad Sci USA 93:3553–3558
54. Zamir I, Harding HP, Atkins GB, Hörlein A, Glass CK, Rosenfeld MG, LazarMA
(1996) A nuclear hormone receptor corepressor mediates transcriptional
silencing by receptors with distinct repression domains. Mol Cell Biol 16:5458–5465
55. Downes M, Burke LJ, Bailey PJ, Muscat GEO (1996) Two receptor interaction
domains In the corepressor, N-Cor/Rip13, are required for an efficient
interaction with Rev-ErbA and RVR: physical association is dependent on the
E region of the orphan receptors. Nucleic Acids Res 24:4379–4386
56. Zamir I, Dawson J, Lavinsky RM, Glass CK, Rosenfeld MG, Lazar MA (1997)
Cloning and characterization of a corepressor and potential component of the
nuclear hormone receptor repression complex. Proc Natl Acad Sci USA
94:14400–14405
57. Zamir I, Zhang JS, Lazar MA (1997) Stoichiometric and steric principles
governing repression by nuclear hormone receptors. Genes Dev 11:835–846
58. Burke LJ, Downes M, Laudet V, Muscat GEO (1998) Identification and
characterization of a novel corepressor interaction region in RVR and
Rev-erbA . Mol Endocrinol 12:248–262
42
LIST OF REFERENCES (continued)
59. Dussault I, Giguère V (1997) Differential regulation of the N-myc proto-oncogene
by ROR and RVR, two orphan members of the superfamily of nuclear
hormone receptors. Mol Cell Biol 17:1860–1867
60. Vu-Dac N, Chopin-Delannoy S, Gervois P, Bonnelye E, Martin G, Fruchart J-C,
Laudet V, Staels B (1998) The nuclear receptors peroxisome proliferatoractivated receptor and Rev-erb mediate the species-specific regulation of
apolipoprotein A-I expression by fibrates. J Biol Chem 273:25713–25720
61. Downes M, Carozzi AJ, Muscat GEO (1995) Constitutive expression of the orphan
receptor, Rev-erbA , inhibits muscle differentiation and abrogates the
expression of the myoD gene family. Mol Endocrinol 9:1666–1678
62. Burke L, Downes M, Carozzi A, Giguère V, Muscat GE (1996) Transcriptional
repression by the orphan steroid receptor RVR/Rev-erbß is dependent on the
signature motif and helix 5 in the E region: functional evidence for a biological
role of RVR in myogenesis. Nucleic Acids Res 24:3481–3489
63. Escriva H, Safi R, Hänni C, Langlois M-C, Saumitou-Laprade P, Stehelin D,
Capron A, Pierce R, Laudet V (1997) Ligand binding was acquired during
evolution of nuclear receptors. Proc Natl Acad Sci USA 94:6803–6808
64. Willy PJ, Mangelsdorf DJ (1998) Nuclear orphan receptors: the search for novel
ligands and signaling pathways. In: O’Malley BW (ed) Hormones and
Signaling. Academic Press, San Diego, CA, pp 307–358
65. Blumberg B, Evans RM (1998) Orphan nuclear receptors-new ligands and new
possibilities. Genes Dev 12:3149–3155
66. Kliewer SA, Lehmann JM, Willson TM (1999) Orphan nuclear receptors: shifting
endocrinology into reverse. Science 284:757–760
67. Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM (1990) Nuclear receptor that
identifies a novel retinoic acid response pathway. Nature 345:224–229
68. Hamada K, Gleason SL, Levi B-Z, Hirschfeld S, Appella E, Ozato K (1989)
H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds
to both regulatory element of major histocompatibility class I genes and the
estrogen response element. Proc Natl Acad Sci USA 86:8289–8293
69. Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro AE,
Kakizuka A, Evans RM (1992) Characterization of three RXR genes that mediate
the action of 9-cis retinoic acid. Genes Dev 6:329–344
43
LIST OF REFERENCES (continued)
70. Leid M, Kastner P, Lyons R, Nakshari H, Saunders M, Zacharewski T, Chen J-Y,
Staub A, Garnier J-M, Mader S, Chambon P (1992) Purification, cloning, and
RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to
bind target sequences efficiently. Cell 68:377–395
71. Oro AE, McKeown M, Evans RM (1990) Relationship between the product of the
Drosophila ultraspiracle locus and the vertebrate retinoid X receptor. Nature
347:298–301
72. Henrich VC, Sliter TJ, Labahn DB, MacIntyre A, Gilbert LI (1990) A
steroid/thyroid hormone receptor superfamily member in Drosophila
melanogaster that shares extensive sequence similarity with a mammalian
homologue. Nucleic Acids Res 18:4143–4148.
73. Shea MJ, King DL, Conboy MJ, Mariani BD, Kafatos FC (1990) Proteins that bind
to Drosophila chorion cis-regulatory elements: a new C2H2 zinc finger protein
and a C2C2 steroid receptor-like component. Genes Dev 4:1128–1140
74. Siu Q, Linney E (1993) The mouse retinoid-X receptor- gene: evidence for
functional isoforms. Mol Endocrinol 7:651–658
75. DLollé P, Fraulob V, Kastner P, Chambon P (1994) Developmental expression of
murine retinoid X receptor (RXR) genes. Mech Dev 45:91–104
76. Dagata T, Kanno Y, Ozato K, Taketo M (1994) The mouse RXRß gene encoding
RXRß: genomic organization and two mRNA isoforms generated by alternative
splicing of transcripts initiated from CpG island promoters. Gene 142:183–189
77. Nevin AA, Sturzenbecker LJ, Kazmer S, Bosakowski T, Huselton C, Allenby G,
Speck J, Kratzeisen C, Rosenberger M, Lovey A, Grippo JF (1992) 9-cis Retinoic
acid stereoisomer binds and activates the nuclear receptor RXR . Nature
355:359–361
78. Leyman RA, Mangelsdorf DJ, Dyck JA, Stein RB, Eichele G, Evans RM,
Thaller C (1992) 9-cis Retinoic acid is a high affinity ligand for the retinoid X
receptor. Cell 68:397–406
79. Hitareewan S, Burka LT, Tomer KB, Parker CE, Deterding LJ, Stevens RD,
Forman BM, Mais DE, Heyman RA, McMorris T, Weinberger C (1996) Phytol
metabolites are circulating dietary factors that activate the nuclear receptor
RXR. Mol Biol Cell 7:1153–1166
80. HeMotte PK, Keidel S, Apfel CM (1996) Phytanic acid is a retinoid X receptor
ligand. Eur J Biochem 236:328–333
44
LIST OF REFERENCES (continued)
81. Lehmann JM, Jong L, Fanjul A, Cameron JF, Lu XP, Haefner P, Dawson MI,
Pfahl M (1992) Retinoids selective for retinoid X receptor response pathways.
Science 258:1944–1946
82. Heinzer, C, Susens, U, Schmitz, TP, Borgmeyer, U (1998) Retinoids induce
differential expression and DNA binding of the mouse germ cell nuclear factor in
P19 embryonal carcinoma cells. Biol Chem 379:349
83. Escriva H, Safi R, Hänni C, Langlois MC, Saumitou-Laprade P,Stehelin D, Capron
A, Pierce R, Laudet V (1997) Ligand bindingwas acquired during evolution of
nuclear receptors. Proc Natl Acad Sci USA 94:6803–6808
84. Rosenfeld, JM, Kao, H-Y, Evans, RM (2003). The nuclear receptor superfamily.
In: “Hormone, Genes, and Cancer”, Henderson, BE, Ponder, B, Ross, RK (eds).
Oxford University Press, New Tork, NY, pp 38-98.
85. Chung, A CK, Katz, D, Pereira, FA, Jackson, KJ, DeMayo, FJ, Cooney, AJ,
O’Malley, BW (2001) Loss of orphan receptor germ cell nuclear factor function
results in ectopic development of the tailbud and a novel posterior truncation.
Mol Cell Biol 21:663-677.
86. Danielsen M, Hinck L, Ringold GM (1989) Two amino acidswithin the knuckle of
the first zinc finger specify DNA Response element activation by the glucocorticoid
receptor. Cell 57:1131–1138
87. Chen F, Cooney AJ, Wang Y, Law SW, O’Malley BW (1994) Cloning of a novel
orphan receptor (GCNF) expressed during germ cell development. Mol Endocrinol
8:1434–1444
88. Hirose T, O’Brien DA, Jetten AM (1995) RTR: a new member of the nuclear receptor
superfamily that is highly expressed in murine testis. Gene 152:247–251
89. Barreto, G, Borgmeyer, U, Dreyer, C (2003) The germ cell nuclear factor is required
for retinoic acd signaling during Xenopus development. Mech Dev 120:415-428.
90. Gu, P, LeMenuet, D, Chung, A C-K, Mancini, M, Wheeler, DA Cooney, AJ
(2005) Orphan nuclear receptor GCNF is required for the repression of
pluripotency genes during retinoic acid-induced embryonic stem cell
differentiation. Mol Cell Biol 25:8507-8519.
91. Pesce, M, Scholer, HR (2001) Oct-4: gatekeeper in the beginnings of mammalian
development. Stem Cells 19:271-278
45
LIST OF REFERENCES (continued)
92. Okamoto, K, Okazawa, H, Okuda, A, Sakai, M, Muramatsu, M, Hamada, H
(1990) A novel octamer binding transcription factor is differentially expressed
in mouse embryonic cells. Cell 60:461-472.
93. Nichols, J, Zevnik, B, Anastassiadis, K, Niwa, H, Klewe-Nebenius, D. Chambers,
I, Scholer, H, Smith, A (1998) Formation of pluripotent stem cells in the
mammalian embryo depends on the POU transcription factor Oct-4. Cell 95:379-391.
94. Shimazaki, T, Okazawa, H, Fujii, H, Ikeda, M, Tamai, K, McKay, RDG,
Muramatsu, M, Hamada, H (1993). Hybrid cell extinction and re-expression of
Oct-3 function correlates with differentiation potential. EMBO J 12:4489-4498
95. Romano, G (2006) The role of adult stem cells in carcinogenesis. Drug News
Perspect 18:555-559 28. Guo,W. Lasky, JL, Wu, H (2006) Cancer stem cells.
Pediatr Res 59:59R-64R
96. Polyak, K, Hahn, WC (2006) Roots and stems: stem cells and cancer.
Nat Med 12: 296-300
97. Niwa, H, Miyazaki, J-I, Smith, AG (2000) Quantitative expression of Oct-3/4
defines differentiation, dedifferentiation or self-renewal of ES cells.
Nature Gen 24:372-376
98. Hochedlinger, K, Yamada, Y, Beard, C, Jaenisch, R (2005) Ectopic Expression
of Oct-4 blocks progenitor cell differentiation and causes dysplasia in epithelial
tissues. Cell 121:465-477
99. Furhmann, G, Chung, AC, Jackson, KJ, Hummelke, G, Baniahand, A, Sutter,
J, Sylvester, I, Scholer, HR, Cooney, AJ (2001) Mouse germline restriction of
Oct-4 expression by germ cell nuclear factor. Dev Cell 1:377-387.
100. Susens, U, Borgmeyer, U (2000) Genomic structure of the gene for mouse germ
cell nuclear factor (GCNF). Genome Biol 1: Research 6.1-6.3
101. Susens, U, Borgmeyer, U (2001) Genomic structure of the gene for mouse germ
cell nuclear factor (GCNF). II. Comparison with the genomic structure of the
human GCNF gene. Genome Biol 2: Research 17.1-17.7
102. Gu, P, Morgan, DH, Sattar, M, Xu, X, Wagner, R, Ravisconi, M,
Lichtarge, O, Cooney, AJ (2005).Evolutionary tree based peptides identify a
novel asymmetric interaction that mediates oligomerization in nuclear
receptors. J Biol Chem 280:31818-31829.
46
LIST OF REFERENCES (continued)
103. Sato, N. Kondo, M, Arai, K-I (2006) The orphan nuclear receptor GCNF
recruits DNA methyltransferase for Oct-3/4 silencing. Biochem Biophys Res
Comm 344:845-851.
104. Moses HL, Pietenpol JA, Munger K,Murphy CS (1991) TGF beta regulation of
epithelial cell proliferation: role of tumor suppressor genes.
Princess Takamatsu Symp. 22:183-95.
105. Salbert G, Fanjul A, Piedrafita FJ, Lu XP, Kim SJ, Tran P, Pfahl M (1993)
Retinoic acid receptors and retinoid X receptor-alpha down-regulate the
transforming growth factor-beta 1 promoter by antagonizing AP-1 activity.
Mol Endocrinol. Oct; 7(10):1347-56.
106. Weis KE, Ekena K, Thomas JA, Lazennec G, Katzenellenbogen BS (1996)
Constitutively active human estrogen receptors containing amino acid substitutions
for tyrosine 537 in the receptorprotein. Mol Endocrinol 10:1388–1398
107. White R, Sjöberg M, Kalkhooven E, Parker MG (1997) Ligand independent
activation of the oestrogen receptor by mutation of a conserved tyrosine. EMBO J
16:1427–1435
108. Vivat V, Zechel C, Wurtz JM, Bourguet W, Kagechika H, UmemiyaH, Shudo K,
Moras D, Gronemeyer H, Chambon P (1997) A mutation mimicking ligandi nduced conformational changeyields a constitutive RXR that senses allosteric
effects in heterodimers. EMBO J 16:5697–5709
109. Bunone G, Briand PA, Miksicek RJ, Picard D (1996) Activation of the unliganded
estrogen receptor by EGF involves the MAP kinase pathway and direct
phosphorylation. EMBO J 15:2174–2183
110. Rochette-Egly C, Adam S, Rossignol M, Egly JM, Chambon P (1997) Stimulation
of RAR alpha activation function AF-1through binding to the general transcription
factor TFIIH and phosphorylation by CDK7. Cell 90:97–107
111. Taneja R, Rochette-Egly C, Plassat JL, Penna L, Gaub MP,Chambon P (1997)
Phosphorylation of activation functions AF-1and AF-2 of RAR alpha and RAR
gamma is indispensable fordifferentiation of F9 cells upon retinoic acid and cAMP
treatment.EMBO J 16:6452–6465
47
APPENDICES
48
Table 1: ES-2 cells were either left untreated or treated with control
GCNF siRNA or GCNF siRNA and were processed for cell counts
72hrs post-transfection. The Data show that GCNF siRNA suppresses
cell proliferation.
S. No
1
2
3
Mean SEM
%
Decrease P Value
relative to
Untreated
Untreated 6858 6646 7244 6916
175.0466 __
__
Control
siRNA
6207 5995 6593 6265
175.0466 9
Ns
siRNA
3642 3430 4208 3760
232.2097 45.6
p<0.001
Table 2: TOV-112 cells were either left untreated or treated with control
GCNF siRNA or GCNF siRNA and were processed for cell counts
72hrs post-transfection. The Data show that GCNF siRNA suppresses
cell proliferation.
S. No
1
2
3
Mean SEM
%Decrease
relative to
P Value
Untreated
Untreated 6206 5994 6592 6264
175.0466 ___
___
Control
siRNA
5645 5433 6031 5703
175.0466 9
ns
siRNA
3889 3677 4275 3947
175.0466 37
p<0.001
49
Table 3: Repeat of Table 2
S. No
1
2
3
Mean SEM
%Decrease
relative to
P Value
Untreated
Untreated 9220 9008 9606 9278
175.0466 ___
___
Control
siRNA
8585 8373 8971 8643
175.0466 6.8
ns
siRNA
5945 5733 6331 6003
175.0466 35.3
p<0.001
50
Table 4: GCNF expression was correlated with cell proliferation by blocking
proliferation with TGF-ß1 which is inhibitory towards MLEC. MLEC were grown in
medium supplemented with 0, 1, or 5 ng/ml TGF-ß1. The impact of TGF-ß1 on cell
proliferation was monitored by cell count analysis. TGF-ß1 dose-dependently inhibited
the MLEC proliferation.
2
3
Mean SEM
% Decrease
relative to
Control
S.NO
1
Control
106358 87087 95754 96400 5572.418 ___
___
1ng
TGF-β
55999
40425 37436 44620 5754.557 53.7
p<0.001
5ng
TGF-β
35648
30467 20976 29030 4295.925 69.9
P<0.001
51
P Value
Table 5: Flasks of cells were processed for RNA isolation and RT/PCR using GCNF
primers following TGF-β treatment. The recorded RNA values are shown here. There
was a concomitant dose-dependent suppression of GCNF mRNA which was confirmed
by RT/PCR.
RNA Values
% Decrease
relative to
control
1
Control
0.2224µg/µl
____
2
1ng TGF-β
0.0515µg/µl
77
3
5ng TGF-β
0.045µg/µl
88
52
Table 6: PA-1 germ cell tumor cells were grown in medium supplemented with 0, 1, or 5
ng/ml TGF-ß. In contrast, TGF-ß1 neither inhibited PA-1 cell proliferation nor GCNF
mRNA expression. The cell count data are as follows
2
3
Mean
S.NO
1
Control
174536 134765 156748 155350 11502.170
___
___
1ngTGF-β
207860 197659 219832 208450 6407.596
34
p<0.05
5ng TGF- β
198437 238543 223754 220245 11709.820
41.8
p<0.01
53
SEM
% Increase
relative to
Control
P values
Figure 1 – GCNF expression in the hamster ovary. Female hamsters at various ages were
terminated and total RNA was prepared from whole ovaries and subjected to RT/PCR
using the GCNF primer set. A predicted 351 bp PCR product was observed at all ages
(left panel). The hamster ovarian GCNF PCR product was subjected to sequence analysis.
The homology between the hamster and mouse (NCBIgi6222570) sequences is illustrated
on the right panel.
54
Figure 2 - Morphology and comparative growth rates of ovarian cancer cell lines in
culture. Appearances of actively growing ES-2, OVCAR-3, TOV-112, and PA-1 cells
shortly after passage (left panel, A-D, respectively). All four photomicrographs are taken
as the same magnification, x 3,600. The solid line in panel A = 1 micron. Growth rates of
ovarian cancer cells in culture (Right panel). ES-2, PA-1, OVCAR and TOV-112 human
ovarian cancer cell were seeded in 24-well plates (25,000-35,000 cells/well) under
conditions specific for each cell type. At 1, 2, 3, and 4 days post inoculation, 4 wells of
cells were counted and the means determined.
55
Figure 3 – Expression of GCNF mRNA and protein by ovarian cancer cell lines.Ovarian
cancer cells were cultured as indicated in the methods section. Total RNA was prepared
from cell monolayers and subjected to RT/PCR utilizing human GCNF primers. All four
cell lines produced a strong band consistent with the predicted 351 bp GCNF PCR
product (Panel A: PA-1, ES-2, and OVCAR-3 cells, lanes 2-4, respectively. Panel B:
PA-1, ES-2, TOV-112, and OVCAR-3 cells, lanes 2-5, respectively). A 100 bp DNA
ladder is contained in lane 1 in both panels A and B and the 500 bp standard indicated by
an arrow. Western blot analysis of GCNF protein from whole cell lysates from: ES-2
(lane 1), TOV-112 (lane 2), and PA-1 (lane 3) cells (Panels C and D). Lysates were run
using non- reducing, polyacrylamide gel electrophoresis. Bands corresponding
toimmunoreactive GCNF were detected using a GCNF antibody directed against the
ligand binding domain (Panel C). The antibody was kindly provided by Dr.Austin
Cooney, Baylor College of Medicine. Non-specific GCNF binding was assessed using
non-immune IgG (Panel D).
56
Figure 4 – The exon structure of GCNF and the location of oligonucleotide Primers (left
panel). The PCR product spanned most of exons 4 and 6 and all of exon 5. The predicted
bands corresponding to GCNF were extracted from agarose gels following RT/PCR of
TOV-112 total RNA and forwarded to an outside lab for sequence analysis. TOV-112
cells were utilized as representative of the 4 cell lines. The TOV-112 cell PCR product
sequence exhibited 100% homology to the published human GCNF sequences in
Genbank (gi53729326) (right panel).
57
Figure 5 – Expression of GDF-9 and Oct-4 mRNA in ovarian cancer cells.
Total RNA was prepared from the ovarian cancer cell lines and subjected to
RT/PCR using oligonucleotide primers. GDF-9 mRNA expression by PA-1, ES-2,
OVCAR cells, and a 11 day old hamster ovary, lanes 2-5, respectively is illustrated
in Panel A. Oct-4 mRNA expression in ES-2, PA-1, TOV-112, OVCAR-3 cells,
lanes 2-5, respectively, is shown in Panel B. Panels C and D illustrate GCNF,
Oct-4, FSH-Rc, and GDF-9 Expression (lanes 2-5, respectively) in TOV-112D and
ES-2 cell. For all panels, lane one represents a 100 bp DNA ladder with specific
markers indicated. For Panels C and D, human-specific primers were used for PCR
analyses of GCNF (187 bp), Oct-4 (312 bp), FSH-Rc (266 bp), and GDF-9
(332 bp), lanes 2-5 respectively.
58
Figure 6 - Impact of all trans-retinoic acid (RA) upon GCNF and Oct-4 mRNA
expression. Sub-confluent ES-2 and TOV-112 cells were either unexposed
(lane 2) or exposed to 0.1% DMSO alone (lane 3) or DMSO containing 10-5 M RA
(lane 4) or 10-6 M RA (lane 5) for 48 hours, whereupon total cellular RNA was
isolated. GCNF and Oct-4 mRNA content as assessed by RT/PCR is shown in the
left and right panels respectively.
59
Figure 7- If GCNF is central to cancer cell proliferation, it follows that suppression
of GCNF expression should decrease proliferation. TOV-112D cells were treated with
control (non-specific) siRNA, GCNF siRNA, or were left untreated. Total cell RNA
was isolated 72 hr post treatment and subjected to RT/PCR for GCNF. Other treated
monolayers were also processed for cell counts using Coulter Counter. Relative to
untreated cells, control si-RNA affected neither GCNF mRNA levels nor cell
proliferation. In marked contrast, GCNF-siRNA reduced GCNF mRNA expression by
37.6% in TOV-112 cells (p<0.05), and actual cell proliferation by 36.2% (p<0.001).
These results strongly implicate GCNF as having a causative role in the proliferation
of ovarian cancer cells.
60
Figure 8- We sought to correlate GCNF expression with cell proliferation by blocking
proliferation with TGF-ß1, which is known to potently inhibit MLEC proliferation.
PA-1(Germ cell tumor cells) and MLEC were grown in medium supplemented with 0,
1, or 5ng/ml TGF-ß1. After 48 hr of culture, flasks of cells were processed for RNA
isolation and RT/PCR for GCNF. In addition, the impact of TGF-ß1 on cell
proliferation was monitored by cell count analysis. TGF-ß1 dose-dependently inhibited
proliferation of the sensitive MLEC as expected by 69.9% (p<0.001). Concomitant
with this, there was a dose-dependent suppression of GCNF mRNA. In contrast,
TGF-ß1 did not inhibit PA-1 cell proliferation nor GCNF mRNA expression.
1HLZ_A: gi 17942647 ([Homo sapiens] Chain A, Crystal
61
Structure Of The Orphan Nuclear Receptor Rev- Erb(Alpha)
DNA-Binding Domain Bound To Its Cognate Response Element)
1HLZ_A
1 ~~~~~~~~~tklngmvllckvcgDVASGFHYGVLACEGCKGFFRRSIQQNIQYKrCLKNE 51
query
1 ~~~~~~~~~~~~~~~~~~~~~~~DRATGLHYGIISCEGCKGFFKRSICNKRVYR~CSRDK 36
gi 1054912 69 qaqmmakpgmtsprpielcavcgDKASGRHYGAISCEGCKGFFKRSIRKHLGYT~CRGNK 127
gi 3127936 12 lsdkirnnsnnlcltvelcvvcgDRASGRHYGAISCEGCKGFFKRSIRKQLGYQ~CRGSK 70
gi 31229063 421 gtgsaddmppddddqplvcmiceDKATGLHYGIITCEGCKGFFKRTVQNRRVYT~CVADG 479
1HLZ_A
52 NCSIVRINRNRCQQCRFKKCLSVGMSRDavrfgripkrekqrm~~~~~~~~~~~~~~~~~ 94
query
37 NCVMSRKQRNRCQYCRLLKCLQMGMNRKairedgmpggrnksigpvqiseeeierimsgq 96
gi 1054912 128 DCQIIKHNRNRCQYCRLQKCLDMGMKSDsvqcersplktrdktpgncaastdkiyirkdi 187
gi 3127936 71 NCEVTKHHRNRCQYCRLQKCLACGMRSDsvqherkpiidkkdysnnignnynsnsvnkif 130
gi 31229063 480 TCEITKAQRNRCQYCRFKKCIEQGMVLQavredrmpggrnsgavynlykvkykkhkksnl 539
Figure 9- c4 zinc finger in nuclear hormone receptors; Structure summary: PDB1HLZ
(MMDB 17905)1HLZ_A: gi 17942647 ([Homo sapiens] Chain A, Crystal structure of
the orphan nuclear receptor Rev- Erb(Alpha) DNA-binding domain bound to its cognate
response element. 1HLZ_B: gi 17942648; 1HLZ_C: gi 17942649; 1HLZ_D: gi
17942650; Heterogens: ZN (x4)
1HG4_A: gi 13399655 ([Drosophila melanogaster] Chain A,
62
Ultraspiracle Ligand Binding Domain from Drosophila.
1HG4_A
109 gagggggglghdgsferrspglqpqQLFLNQSFSYHRNSAI~~~~~~~~~~~~~~~~~~~ 149
query
278 ~~~~~~~~~~~~~~~~~~~~~~~~~GELADVTAKYSPSDEElh~~~~~~~~~~~~~~~~~ 295
gi 12643728 342 ~~~~~~~~~~~~~~~~~~~~~~~~~GELADVTAKYSPSDEElh~~~~~~~~~~~~~~~~~ 359
gi 3913096 268 ~~~~~~~~~~~~~~~~~~~~~~~~~PLLAAAGLHASPMSAD~~~~~~~~~~~~~~~~~~~ 283
gi 3093388 660 ~~~~~~~~~~~~~~~~~~~~~~~~~DQLTTVIKTDFNHEVEtnlctlqscltsmmgreit 69
1HG4_A
150 ~~~KAGVSAIFDRILSELSVKMKRLNLDRRELSCLKAIILYNPDIRGIKS~~RaeIEMCR 204
query
296 ~~rFSDEGMEVIERLIYLYHKFHQLKVSNEEYACMKAINFLNQDIRGLTSasQ~~LEQLN 351
gi 12643728 360 ~~rFSDEGMEVIERLIYLYHKFHQLKVSNEEYACMKAINFLNQDIRGLTS~~AsqLEQLN 415
gi 3913096 284 ~~~RVVAFMDHIRIFQEQVEKLKALHVDSAEYSCLKAIVLFTSDACGLSD~~VahVESLQ 338
gi 3093388 695 ieqLRQDVGLMIEKITHVTLMFRQIKLTMEEYVCLKVITMLNQAKPASSSg~NseLESIH 753
1HG4_A
205 EKVYACLDEHCRLEHpGDDGRFAQLLLRLPALRSISlkcqdhlflfritsdrpleelfle 264
63
query
352 KRYWYICQDFTEYKYtHQPNRFPDLMMCLPEIRYIAgkmvnvpleqlpllfkvvlhsckt 411
gi 12643728416 KRYWYICQDFTEYKYtHQPNRFPDLMMCLPEIRYIAgkmvnvpleqlpllfkvvlhsckt 475
gi 3913096 339 EKSQCALEEYVRSQYpNQPTRFGKLLLRLPSLRTVSssvieqlffvrlvgktpietlird 398
gi 3093388 754 ERYMTCLRVYTQHMYpQQTTRFQDLLGRLPEIQSAAfllleskmfyvpfllnsaiqr~~~ 810
Figure 10- Ligand binding domain of hormone receptors; Structure summary: PDB
1HG4 (MMDB 15636)1HG4_A: gi 13399655 ([Drosophila melanogaster] Chain A
Ultraspiracle Ligand Binding Domain from Drosophila Melanogaster); 1HG4_B: gi
13399656; 1HG4_C: gi 13399657; 1HG4_D: gi 13399658; 1HG4_E: gi 13399659;
1HG4_F: gi 1339966; Heterogens: LPP (x6)
64