Mechanistic Investigation of Anticancer Agents that Damage
DNA and Interact with the Estrogen Receptor
By
Sreeja Gopal
B.Tech. Chemical Engineering (2003)
Indian Institute of Technology, Madras
SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL ENGINEERING IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
ARCHIVES
DOCTOR OF PHILOSOPHY IN BIOLOGICAL ENGINEERING
MASSACHUSET-S INSTITUTE-
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
L-N3(
IL"I
JUNE 2009
@2009 Sreeja Gopal. All rights reserved.
The author hereby grants to MIT permission to reproduce and to
distribute publicly paper and electronic copies of this thesis document in
whole or in part in any medium now known or hereafter created.
Signature of Author:
Departmedt of Biological Engineering
May 22, 2009
Certified by:
John M. Essigmann
William and
sy Leitch Professor of Chemistry and Toxicology
Thesis Supervisor
Accepted by:
-
/Alan
J. Grodzinsky
Biological Engineering
Professor of EVctrical, Mech nical,:
hair, Course XX Graduate Program Committee
EI2F?
THIS DOCTORAL THESIS HAS BEEN EXAMINED BY A COMMITTEE OF THE
DEPARTMENT OF BIOLOGICAL ENGINEERING AS FOLLOWS:
A
Peter C. Dedon
Professor of Toxicology dnd Biological Engineering
Chairman
John M.
Essigma
William and Betsy Leitch Phge sor of Chehtry and Toxicology
Thesis Supervisor
Ram Sasisekharan .
Professor of Biological Engineering and Health Sciences & Technology
Robert G. Croy
_
_
_r
Department of Biological Engineering
(i --.,
Mechanistic Investigation of Anticancer Agents that Damage DNA
and Interact with the Estrogen Receptor
by
Sreeja Gopal
Submitted to the Department of Biological Engineering on May 22, 2009
in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in
Biological Engineering
ABSTRACT
One of the primary goals of cancer chemotherapy is the design of antitumor agents that
achieve selective targeting of tumor cells while minimizing toxicity to normal tissues. We
have synthesized a series of DNA damaging agents that are designed to disrupt
selectively the DNA repair and signaling pathways in tumor cells. My dissertation
focuses on the mechanistic studies of the rationally designed genotoxicants E2-7aX and
2Pl, which target cancer cells that express the estrogen receptor (ER). The studies on
the biological activity of E2-7ca in ovarian cancer cells reveal that the compound induces
levels of cytotoxicity comparable to those of cisplatin, which is the current front-line
therapeutic for ovarian cancer. E2-7ca induces crosslink formation in the ovarian cell line
SKOV-3, which potentially leads to the S-phase arrest observed in these cells. Further,
the arrest is persistent even after drug removal, indicating that the persistence might be
one of the reasons for the enhanced toxicity of E2-7ca compared to the control compound
chlorambucil. E2-7ca induces cell death in SKOV-3 cells through autophagy, as opposed
to the classical cell death pathway of apoptosis. Mechanistic studies on the role of ER in
E2-7a toxicity in SKOV-3 cells show that the absence of the ER (facilitated by the
knockdown of ER protein through RNA interference in SKOV-3 cells) makes the cells
less sensitive to the drug. In addition, the ER(-) population displays a lower level of drugDNA adducts than its ER(+) counterpart, as detected by Accelerator Mass Spectrometry
(AMS). The combination of these pieces of evidence strongly suggests that as per its
intended mechanism of action, E2-7a. mediates its effects through the involvement of the
ER, and appears to be a promising candidate as an antitumor agent in the clinical setting.
In addition, the toxicity and DNA damage caused by the 2-phenylindole compounds 2PI
and 2PI(OH) were studied in ovarian cancer cell lines; these studies underscore the
differences in the mechanism of action of the two compounds. Both the compounds
demonstrate promise as potent anti-tumor agents, and the selectivity of these
compounds in different cellular contexts (ER (+)/(-) or p53 (+)/(-)), once established,
could help strengthen the basis of their therapeutic efficacy.
Thesis Supervisor: John M. Essigmann
Title: William and Betsy Leitch Professor of Chemistry and Toxicology
ACKNOWLEDGMENTS
To Professor John Essigmann, for his invaluable support and guidance. I have been
inspired by his outlook, both towards science and towards life. I am grateful to him for
having provided an environment conducive for intellectual growth and collaborative
learning in his lab. John's passion for teaching and his zest for perfection in oral and
written presentations have helped hone my skills in this regard as well. I thank John for
providing the opportunity to learn from him and work with him.
To my thesis committee members, Professor Pete Dedon, Professor Ram Sasisekharan,
and Dr. Bob Croy, for their advice and insight. I appreciate the time each of you has put
in for regular meetings, and for valuable suggestions to address several aspects of my
research.
To Dr. Bob Croy, who has been amazing as a mentor and advisor to my project. His
extensive knowledge in the field and his guidance with experimental design and
troubleshooting have been crucial for my progress in this project. I really appreciate his
time and his willingness to spend time on my research.
To the members of the Essigmann laboratory, for being great colleagues and friends
over the past five years. A special thanks to Kyle Proffitt, my first teacher in the lab.
Thanks Kyle, for showing me the ropes in setting up experiments and for teaching me
how to do tissue culture and westerns. Your tenacity and your exhaustive approach to
problem solving have been an inspiration. I also wish to thank John Marquis, Shawn
Hillier and Pei-Sze Ng for our collaborative efforts, and Jeannette Fiala and Leslie Woo
for their wonderful jobs editing my thesis drafts. Our seamless interactions at the
Essigmann lab are much appreciated and have made my graduate career so much more
enjoyable. I also thank Kim Schaefer, for her willingness to give administrative
assistance, and for helping things run smoothly in the lab. The Essigmann lab has been
a close-knit community, and I will definitely miss our interactions and our conversations
on both research and on life. I also wish to thank Rosa Liberman and Paul Skipper at the
Tannenbaum laboratory for our collaborations on the AMS front.
To my fellow BE graduate students, for being great friends, and for making my transition
to the US an enjoyable experience. The fun, the conversations, and the mutual support
have been truly memorable.
To my friends, both at MIT and outside, for believing in me, and for listening to me even
when I go on endlessly about the woes of research. I really appreciate your support and
your encouragement.
Most importantly, to my family - my parents, my sister, my grandparents and my
husband - for always believing in me, for inspiring me to be a better person, and for
encouraging me in all my endeavors. Now that my journey here is almost drawing to a
close, it is as much your achievement as it is mine. I forever will appreciate all the time
you have put in on my behalf and your unconditional love and support. What I am today,
I owe to you.
Dedicatedto my lovingfamily
This work would not have been possible without your constant support,
encouragement and love
TABLE OF CONTENTS
COMMITTEE PAGE
2
ABSTRACT
3
ACKNOWLEDGEMENTS
4
TABLE OF CONTENTS
7
LIST OF ABBREVIATIONS
9
LIST OF TABLES
11
LIST OF FIGURES
12
CHAPTER 1: Introduction
15
1.1
1.2
CANCER .....................................................................................
. . 16
1.1.1
Cancer Therapy and Therapeutics
19
1.1.2
Ovarian Cancer
21
1.1.3
Current Therapy for Ovarian Cancer
23
26
NUCLEAR HORMONE RECEPTORS ..................................................
1.2.1
27
Estrogen Receptor
31
1.3
ER AND OVARIAN CANCER .............................................................
1.3
RATIONAL DESIGN OF NOVEL GENOTOXICANTS THAT TARGET ER(+)
T UMO RS ..........................................................................................
33
1.3.1
E2-7a-C6NC2-Mustard
34
1.3.2
Phenylindole-C6NC2-Mustard
35
1.4
FIG UR ES .....................................................................................
1.5
REFER EN CES ................................................................................
. . 37
42
CHAPTER 2: E2-7a - Biological Consequences in Ovarian Cancer Cells:
Cytotoxicity, DNA Damage and Cell Death
2.1
IN T R O DUCT IO N ...................
.............
7
59
............................................ 60
2.2
MATERIALS AND METHODS ...........................................................
2 .3
RESULT S .......................................................................................
2.4
DISCUSSION .........
2.5
CONCLUSIONS ........................................
2 .6
T AB L ES ............................................................................................
91
2.7
FIGURES ........................................................................................
93
2.8
REFERENCES ............................................
.....................................
68
. 73
80
....89
107
CHAPTER 3: Role of ER in E2-7a toxicity: Studies in ER(+) and ER(-)
cell
populations
139
3.1
INTRODUCTION
3.2
MATERIALS AND METHODS .............................................................
146
3 .3
RESULT S ........................................................................................
14 9
3.4
DISCUSSION ...................................................................................
153
3.5
CONCLUSIONS ...........................................
159
3.6
FUTURE DIRECTIONS ......................................................................
160
3 .7
TA B LES ..........................................................................................
16 2
3 .8
FIG URES .........................................................................................
16 4
3.9
REFERENCES ............................................
170
........................
.........
................... 140
CHAPTER 4: Investigation of cytotoxicity and other biological properties of
phenylindole compounds in ovarian cancer cells
203
4.1
INTRODUCTION ................................................................................
204
4.2
MATERIALS AND METHODS .............................................................
206
4 .3
RESUL TS ........................................................................................
2 11
4.4
DISCUSSION ................
.....
4.5
CONCLUSIONS .............
...................
4.6
TABLES .......................................................................................
225
4.7
FIG URES .........................................................................................
226
4.8
REFERENCES
................
... ...............................
......
....................... 217
........................................... 224
......................................... 237
LIST OF ABBREVIATIONS
AF
Activation Function
AMS
Accelerator Mass Spectrometry
BRCA1/ BRCA2
Breast Cancer 1/2
CA-1 25
Cancer Antigen 125
Cdk
Cyclin-dependent kinase
CDT
Charcoal Dextran-treated
cpm
Counts per minute
DBD
DNA Binding Domain
dpm
Disintegrations per minute
DMSO
Dimethyl Sulphoxide
E2-7a
Estradriol-C6NC2-mustard
EB
Ethidium Bromide
EOC
Epithelial Ovarian Cancer
ER
Estrogen Receptor
ER-LBD
Estrogen Receptor Ligand Binding Domain
ERE
Estrogen Response Element
ESI-MS
Electrospray Ionization Mass Spectrometry
HPLC
High Performance Liquid Chromatography
HNPCC
Hereditary Non-Polyposis Colon Cancer
HRE
Hormone Response Element
HRT
Hormone Response Therapy
HSP
Heat Shock Protein
ICL
Interstrand Crosslink
LBD
Ligand Binding Domain
MDC
monodansylcadaverine
MEM
Minimum Essential Medium
NLS
Nuclear Localization Signal
NER
Nucleotide Excision Repair
NHR
Nuclear Hormone Receptor
OC
Ovarian Cancer
OSE
Ovarian Surface Epithelium
OTM
Olive Tail Moment
PBS
Phosphate Buffered Saline
2PI
2-Phenylindole
PR
Progesterone Receptor
SDS
Sodium Dodecyl Sulphate
siRNA
Short interfering RNA
XPA
Xeroderma Pigmentosum group A
LIST OF TABLES
Table 2.1
Table 2.2
G150 values of test compounds in SKOV-3 and OVCAR-3
ce lls ..........................................................................
. . 91
Comet assay detects E2-7x induced crosslinks in SKOV-3
ce lls ..........................................................................
. . 92
Table 3.1
Doubling times of SKOV-3 cells with estrogen and antiestrogen
tre atm e nt........................................................................16 2
Table 3.2
Adducts, cell numbers, and [14C]-E2-7a distributions in ER(+) and
ER(-) SKO V-3 cells...........................................................163
Table 4.1
Percentage decrease in OTM in Caov-3 and OVCAR-3 cells.....225
LIST OF FIGURES
Figure 1.1
Structures of Relevant Chemotherapeutic Agents................. 37
Figure 1.2
Structural Organization of Nuclear Hormone Receptors and
Estrogen Receptors a and P..............................................38
Figure 1.3
Possible Mechanisms of Action of Bifunctional Genotoxicants.....39
Figure 1.4 A Novel Agent Designed to Inhibit Repair Processes in a Cancer
Cell ............................................................................
. . 40
Figure 1.5
Structures of Bifunctional Genotoxicants that Target the ER........41
Figure 2.1
Mechanism of Reaction of E2-7a with DNA...........................93
Figure 2.2
E2-7a Treatment is Cytotoxic to SKOV-3 Cells following 24 and 36
Hours of Treatm ent........................................................ 94
Figure 2.3
E2-7a Treatment is Cytotoxic to OVCAR-3 Cells following 24 and
36 Hours of Treatment....................................................
95
Figure 2.4
E2-7ax Arrests SKOV-3 Cells in S-Phase.............................96
Figure 2.5
E2-7a Arrests OVCAR-3 Cells in S-Phase...........................97
Figure 2.6
Chlorambucil Treated Cells Recover from Arrest once Drug is
Rem oved ....................................................................
. . 98
Figure 2.7
E2-7a Treated Cells Do Not Recover from Arrest once Drug is
Rem oved ....................................................................
. . 99
Figure 2.8
Figure 2.9
E2-7a Induces Activation of Checkpoint Proteins in SKOV-3
cells ...............................................................................
100
Comet Assay Indicates Presence of Interstrand Crosslinks after
E2-7c Treatm ent..............................................................101
Figure 2.10 XPA cells are more sensitive to E2-7c than HeLa cells.............102
Figure 2.11 Apoptotic markers are not activated with E2-7a treatment.......103
Figure 2.12 AO/EB Staining of Live SKOV-3 Cells (5 pM, 12 hours)............104
Figure 2.13 AO/EB Staining of Live SKOV-3 Cells (2.5 pM, 24 hours)..........105
Figure 2.14
MDC Staining of Live SKOV-3 Cells (2.5 pM, 24 hours)............106
Figure 3.1
SKOV-3 Cells are not Growth Sensitive to Estradiol or ICI
18 2,780..........................................................................
16 4
Figure 3.2
ER is Downregulated with siRNA Treatment for up to 72 Hours..165
Figure 3.3
ER(+) and ER(-) SKOV-3 Cells are Equally Sensitive to Melphalan.
They are Equally Sensitive to Cisplatin as well ........................ 166
Figure 3.4
ER(+) Cells are More Sensitive to E2-7a than ER(-) Cells.......167
Figure 3.5
E2-7a Forms DNA Adducts in SKOV-3 Cells..........................168
Figure 3.6
E2-7a Adduct Levels are Higher in ER(+) compared to ER(-)
SKOV-3 Cells..................................................................169
Figure 4.1
Synthetic Scheme of 2-Phenylindole Compounds....................226
Figure 4.2
Structures and Binding Affinities of Phenylindole Compounds... .228
Figure 4.3
ER Status of Relevant Cell Lines.........................................229
Figure 4.4
2PI and 2PI(OH) Cause Growth Inhibition of Caov-3 and OVCAR-3
cells ...............................................................................
230
Figure 4.5
Cell Cycle Distribution in Caov-3 Cells Treated with Test
Compo unds.....................................................................2 31
Figure 4.6
Cell Cycle Distribution in OVCAR-3 Cells Treated with Test
Compo unds .....................................................................
232
Figure 4.7
Detection of Interstrand Crosslinks in Caov-3 Cells Using Comet
A ss ay .............................................................................
233
Figure 4.8
Detection of Interstrand Crosslinks in OVCAR-3 Cells Using Comet
A ssa y .............................................................................
234
Figure 4.9
Drug-DNA Adducts in Caov-3 and OVCAR-3 cells Detected Using
A MS..............................................................................
235
Figure 4.10
Caspase-3 Activation by Cisplatin and Phenylindole
Compounds.....................................................................236
Chapter 1
Introduction
Cancer
Cancer is a complex disease that results from the coordinated dysregulation of cellular
regulatory circuits. It is characterized by compromised genomic integrity and manifests
as excessive proliferation of a group of cells that invades surrounding tissue and in some
cases, metastasizes to other parts of the body. The process of transformation - the
genetic alteration of a normal cell to a cancer cell - occurs when a cell loses the ability to
regulate its growth and proliferative pathways. It is apparent from current studies that a
normal cell, during its life cycle, could progressively accumulate mutations that provide it
certain growth and survival advantages that enable the cell to transform to a cancer cell.
Studies on cancer incidence and its genomic characterization reinforce the theory that
the transformation from a normal cell to a cancer cell is a multi-step sequence of
stochastic events (Armitage and Doll, 1954; Foulds, 1954; Renan, 1993). The idea is
that each of these steps corresponds to a genomic insult that takes the normal cell one
step closer to malignancy (Bishop, 1991). For example, a normal cell could acquire a
mutation in a tumor suppressor gene that confers it an enhanced rate of proliferation,
and then, in an independent step, acquire the capability to resist apoptosis. Hanahan
and Weinberg (2000) have proposed a framework to explain the steps involved in
cancer progression. There are six characteristic traits of cancer cells that represent the
cellular control mechanisms that normal cells have overcome in their path to becoming
malignant. These traits consist of: (i) self-sufficiency in growth signals, (ii) insensitivity to
anti-growth signals, (iii) ability to evade apoptosis, (iv) limitless replicative potential, (v)
sustained angiogenesis, and (vi) tissue invasion and metastasis. These traits result from
a variety of physical changes in the genome, including somatic mutations, chromosomal
loss and duplication, genomic rearrangements, methylation of DNA, and changes in
chromatin structure. Virtually all cancers display these six traits, but the order or the
mechanism of acquiring these traits is usually not the same in all cells. One mutation
could result in more than one trait at a time, or on the other hand, a single trait might
need two or more coordinated steps to be acquired. These parallel pathways to
malignancy account for the high levels of heterogeneity found amongst the vast array of
cancers.
The susceptibility of a cell towards accumulating mutations is affected by several
endogenous factors, such as levels of DNA repair, immune conditions, hormone levels,
and fidelity of replication. Inherited mutations in key tumor suppressor genes, oncogenes,
and genome maintenance genes are also potential endogenous risk factors. The most
significant causative link to cancer risk comes through the route of exogenous agents,
which include environmental carcinogens, occupational chemicals, radiation and tobacco
smoke, and exposure to pathogens (for example, certain viruses and parasites).
Exposure to these agents can occur through exogenous routes such as air, food, and
water or endogenous processes such as byproducts of inflammation and metabolic
conversion of otherwise innocuous environmental agents.
The existence of such a wide variety of risk factors for cancer could explain the high
number of cases diagnosed each year. According to Cancer Facts and Figures, cancer
is the second leading cause of death in the US and over 500,000 people are expected to
die of cancer this year (American Cancer Society, 2008). Better treatment options over
the last few decades have increased the five-year survival rate among Americans to
66% for all cancers diagnosed between 1996 and 2003, up from 50% in 1975-77. The
numbers of new cases this year of the five most common cancer types - lung cancer
(215,000), prostate cancer (186,000), breast cancer (184,000), colorectal cancer
(148,000) and lymphoma (74,000) - reflect the urgency in investigating more effective
treatment options for the disease.
Cancers diagnosed at earlier stages result in better prognoses, and currently, there are
several options for detecting and treating early stage malignancies. Some cancers can
be screened using specific markers or visual evidence. For instance, elevated level of
Prostate Specific Antigen (PSA) in patients is used as a biomarker for prostate cancer.
Another tumor marker protein Cancer Antigen 125 (CA-1 25) is sometimes used to
screen women with increased risk for ovarian cancer (Bandera et al., 2003; Jacobs and
Bast, Jr., 1989). Mammography is used as a visual tool to assess breast cancer,
sigmoidoscopy to inspect the colon, and Pap test for cervical cancer (Rimer and
Schildkraut, 1997). However, several cancers do not possess well-defined screens to
detect early signs of the disease. Malignancies such as epithelial ovarian cancer are not
detected until the disease is at an advanced stage, and this leads to extremely poor
prognosis for the patient.
Once the cancer is detected and its extent determined, there is a constantly expanding
array of options for therapy. However, it is especially challenging to develop effective
treatment options given the enormous heterogeneity observed in tumor populations the response to treatment can vary significantly depending on the extent of the disease,
its severity and subtype (Rimer and Schildkraut, 1997). The next section discusses
current therapy landscape and the development of newer, improved therapeutics.
Cancer Therapy and Therapeutics
The main goal of cancer therapy is to reduce the tumor cell population to zero cells. This
outcome is best achieved by surgical removal of the tumor to prevent the tumor from
recurring or metastasizing. Surgical resection of the tumor is usually followed by
adjuvant therapy that consists of one or more of treatments such as radiation,
chemotherapy,
immunotherapy,
hormone
therapy,
and/or
bone
marrow
transplantation(Chabner and Longo, 2009). Of these, chemotherapy is historically one of
the oldest known treatments and is often the first choice of therapeutic intervention in
patients with several types of solid tumors and hematopoietic neoplasms (Papac, 2001);
it is almost always used as a systemic treatment and has been shown to cure some
disseminated cancers. Although rarely curative, chemotherapeutics have been effective
in decreasing tumor volume, alleviating symptoms, and even prolonging life in certain
metastatic cancers (Chabner and Longo, 2009; DeVita et al., 1997 ).
Nitrogen mustards - mechlorethamine ("nitrogen mustard", HN2; Figure 1.1) and
tris(p-chloroethyl)amine (HN3) - were the first known chemotherapeutic agents. These
compounds were originally used in chemical warfare in the 1930s; in a serendipitous
turn of events, they were effectively used to treat non-Hodgkin's lymphoma, thus
demonstrating for the first time that chemotherapy could induce tumor regression (Papac,
2001). The clinical use of nitrogen mustards as therapeutic agents was followed by the
use of several other classes of antineoplastic agents such as antifolates, purine analogs,
vinca alkaloids, microtubule inhibitors and topoisomerase inhibitors (Chabner and
Roberts, Jr., 2005). Subsequently, combination chemotherapy and adjuvant therapy
were introduced, and these regimens are now the standard of care treatment of human
malignancy. In fact, combination of drugs have proved to be more effective than single
agents both in metastatic cancer and as adjuvant therapy in cases with a high risk of
relapse (Bonadonna et al., 1976; Jaffe et al., 1974; Li et al., 1958).
The task of development of chemotherapeutics has been arduous - at almost every step,
researchers have encountered significant problems because of long-term and acute
toxicities that affect other organs in the body. A slight respite to this problem came with
the improvements in molecular and genetic understanding of cell biology, which in turn
led to more targeted therapeutics. A variety of compounds, including designed small
molecules, monoclonal antibodies, peptidomimetics, short interfering RNAs (siRNAs),
antisense oligonucleotides and other targeted therapies are being evaluated for the
treatment of cancer (Clark, 2006).
Tools are now available to manipulate molecular systems for therapeutic purposes. The
goal of current therapy development is to target specific proteins that elicit the desired
response from cancer cells while circumventing off-target effects. We at the Essigmann
lab are interested in creating next generation chemotherapeutics by designing
mechanism-based drugs - a series of agents that combine a molecularly targeted
moiety with the proven efficacy of DNA alkylating agents, thus bringing a layer of
selectivity to the DNA alkylating capability of the agent. With this combination, we aim to
augment the efficacy of the antitumor agent by honing in on a selected population of
cancer cells and killing these cells.
DNA damaging agents used as cancer therapeutics are one of the most effective
treatments in clinical use, and have demonstrated improved efficacy when used in
combination with drugs that have a different mechanism of action (Hurley, 2002). A
targeted DNA damaging agent would provide a means of specifically killing malignant
cells while sparing normal tissues. The antitumor agents designed in the Essigmann
laboratory have shown promising results that implicate a component of selectivity in their
toxicity (Mitra et al., 2002; Rink et al., 1996). Understanding the mechanism of action of
these drugs would help assess the potential of these agents as clinically effective
chemotherapeutics. My dissertation is focused on probing the mechanism of action of
two such rationally designed drug candidates that target ovarian cancer.
Ovarian Cancer
Ovarian Cancer (OC) is the leading cause of gynecological death worldwide - there
were 204,000 estimated cases in 2002 and 125,000 deaths due to the disease in the
world (Parkin et al., 2005). According to statistics provided by the American Cancer
Society, 69% of the patients with advanced OC survive but this rate drops to 29% in
patients with distant metastases (American Cancer Society, 2008). OCs can be
subdivided into three major types: surface epithelial-stromal tumors, germ cell tumors
and sex cord-stromal tumors. Of these, epithelial ovarian carcinoma dominate the
cancer registries by accounting for -90% of ovarian tumors in the Western world
(Greenlee et al., 2000). There are approximately 22,000 new cases and 15,000 deaths
associated with this disease every year (Jemal et al., 2008). The relative lack of specific
early signs and symptoms combined with inefficient screening results in overall low cure
rates for epithelial ovarian cancers (EOC).
The etiology of EOC is currently unknown, although there are several risk factors that
have been identified by epidemiological studies. The major risk factors (Yap et al., 2009)
consist of :
(i)
Environmental and hormonal factors: The origin and progression of
ovarian carcinogenesis are still highly debated. Epidemiological studies
show that the risk factors that increase risk are age, infertility, whereas
increasing parity, oral contraceptive use, hysterectomy or tubal ligation
decrease risk (Arai et al., 2004; Riman et al., 2001; Riman et al., 2002b;
Riman et al., 2002a; Riman et al., 2004; Risch, 2002; Schildkraut et al.,
1996 ). One theory regarding the origin of EOC is that frequent ovulations
disrupt the ovarian surface epithelium (OSE) with repeated cycles of
wounding, proliferating, and healing and this may lead to malignant
transformation (Rao and Slotman, 1991). A second cause might be the
persistent stimulation of the ovary by the pituitary gonadotropins luteinizing hormone and follicle-stimulating hormone - which act directly or
mediate their effect through the action of estrogen.
(ii)
Hereditary factors:
Family history of breast or ovarian cancer is a
substantial risk factor for EOC - 10-20% EOCs have a hereditary
predisposition (Pal et al., 2005). Women with BRCA1 or BRCA2 mutations
are at a higher risk, with an average cumulative risk of -40% and -10%
respectively at the age of 70 (Lukanova and Kaaks, 2005). Remarkably
enough, up to 70% of ovarian cancers show mutations in the BRCA1 gene,
in contrast to 20-40% in inherited breast cancers (Merajver et al., 1995; Miki
et al., 1994; Zheng et al., 2001). Early onset of breast cancer, presence of
ovarian cancer, and male breast cancer also point to a hereditary risk for
EOC. Hereditary nonpolyposis colon cancer (HNPCC) is an observed risk
factor in EOC as well (Lindor et al., 2006).
Uk
Current Therapy for Ovarian Cancer
In spite of the knowledge of the risk factors discussed above, in most cases, it is difficult
to detect the disease at an early stage. Since early-stage EOC has the best prognosis
with appropriate treatment, it is indeed desirable to detect signs of the disease early. The
primary marker used for the assessment of the pelvic mass is CA-125 (Bast, Jr. et al.,
1983). Malignancies other than EOC can also upregulate the levels of CA-125, but the
highest levels are usually observed with ovarian cancer. However, only 50% of stage-I
cases were found to have elevated levels of the marker in serum (Jacobs and Bast, Jr.,
1989). Hence, efforts are still ongoing to find more reliable markers and therapies for the
disease, using high throughput techniques such as protein microarrays (Bandera et al.,
2003; Chatterjee et al., 2006).
Accurate diagnosis and stage determination are the first steps in the treatment of women
with EOC. Several clinical trials over the past few decades have made improvements in
treatment of the disease- surgery followed by adjuvant chemotherapy is currently the
standard treatment regimen (Mutch, 2002). Surgical cytoreduction of metastatic disease
is crucial in disease management. Post-surgery, patients who have a significant risk of
recurrence are usually treated with a combination therapy of taxanes and platinumbased drugs (DeVita et al., 1997). While a majority of the patients respond initially to
treatment, most relapse and, unfortunately, in the relapsed population there is only a 2025% five year survival rate for advanced stage EOC (Ozols, 2005). There are two
measures of treatment efficiency - overall survival and progression-free survival (PFS).
Recent studies have shown that chemotherapy has been able to improve PFS statistics
and overall survival in recurrent cases, and therefore, chemotherapeutic regimens have
been used as a benchmark to compare new therapies in Phase Ill trials (Ozols, 2005).
23
Recurrent cases, however, usually develop resistance to chemotherapy, and the
patient's response to the agent decreases with each incidence of relapse after the
development of resistance. Despite the efforts over several years to develop improved
therapeutics, clinical responses have been short-lived, and have only led to marginal
improvements in the treatment of platinum-resistant EOC (Agarwal and Kaye, 2003;
Bookman et al., 2003). Given this therapy landscape, better understanding of the origins
of the disease would be invaluable in developing better therapies.
The view that EOC is an endocrine-related disease has been supported by several
observations. The increase in incidence of EOC with the use of Hormone Replacement
Therapy (HRT) or with the onset of climacterium and the decrease in incidence in
women whose ovulations were suppressed by pregnancy or oral contraceptive use
support the involvement of hormone levels in disease etiology (Argento et al., 2008;
Borini and Rebellato, 2008; Lukanova and Kaaks, 2005; Riman et al., 2004). The
primary hormone involved in the growth and differentiation in normal ovaries is estrogen.
This hormone plays a key role in dictating the physiology of ovarian cells and is also
implicated in the development and progression of ovarian carcinoma, but the mechanism
of estrogen action is unclear. The effectors of estrogen action in cells are estrogen
receptors c and p (ERax and ERp). ERa and ERp are members of the family of nuclear
hormone receptors (also known as steroid receptors) and their transcriptional activity is
mediated by ligand binding and subsequent binding to Estrogen Response Elements
(ERE) in target sequences (Pearce and Jordan, 2004). Studies suggest that ERP, which
is the principal form of ER found in normal OSE, exerts a protective effect against
carcinogenesis whereas ERa, which is the predominant receptor present in ovarian
cancers, promotes tumor progression.
As tumors progress to malignancy, ERp
expression decreases, resulting in ERa becoming the increasingly predominant form
24
found in malignant ovarian tumors (Bardin et al., 2004; Kuiper et al., 1997; Lazennec,
2006; Lindgren et al., 2004; O'Donnell et al., 2005; Rutherford et al., 2000).
The significance of estrogen in the etiology of ovarian cancer is underscored by the
efficacy of estrogen-directed treatments against ovarian cancer. Estrogen has been
shown to induce proliferation in some ER(+) ovarian cancer cells, and antiestrogens
have been shown to inhibit the growth of ovarian carcinoma (Langdon et al., 1993; Nash
et al., 1989). Intervention with aromatase inhibitors has shown some clinical benefits for
EOC patients as well (Krasner, 2007; Rao and Miller, 2005; Walker et al., 2007). As twothirds of EOCs express ERa at the time of diagnosis (Slotman and Rao, 1988), a
promising route to target the endrocrine-dependent EOC is via ERa. Currently, the
treatment options for primary and recurrent cancers are limited, and research focuses on
developing new modalities of selectively targeting ovarian cancer and its metastases.
Several molecular therapies have been developed based on our enhanced
understanding of the disease. Therapies that target characteristics such as angiogenesis,
survival, growth and metastases are now entering clinical trials (Burger, 2007; Hanahan
and Weinberg, 2000). The work I have described in this dissertation focuses on the
development of novel chemotherapeutics that selectively target cancer cells that express
ERa and these drug candidates have been studied in the context of ovarian cancer. I
shall first discuss in detail the role of ERa in ovarian cancer, and then illustrate how the
genotoxicants have been designed to attack cancer cells by exploiting the role of ERa in
the cell.
Nuclear Hormone Receptors
Nuclear Hormone Receptors (NHRs), also known as steroid receptors, are ligandinducible transcription factors that modulate gene expression by binding to specific
recognition sequences in DNA.
They regulate multiple biological functions during
embryonic development and in adult tissues, and they are centrally involved in the
physiological basis of many diseased states (Noy, 2007). Members of the NHR family
include receptors for lipophilic substances such as steroid hormones (estrogen,
progestins, androgens, glucocorticoids, mineralocorticoids, and vitamin D), retinoic acids,
and thyroid hormones (Olefsky, 2001; Parker and Jensen, 1991).
The primary structures of most of the NHRs have been elucidated by sequencing their
cDNAs (Parker, 1991). Such molecular cloning and structure/function analyses have
shown that all NHRs have common structural features, as shown in Figure 1.2A
(adapted from Kumar and Thompson, 1999). As can be seen from the Figure, NHRs
possess a C-terminal Ligand Binding Domain (LBD) and a highly conserved central DNA
Binding Domain (DBD). The LBD is responsible for binding to the signaling ligand and
allows for recruiting coactivator complexes and translocation to the nucleus. In their
unliganded state, nuclear receptors can be found either in the cytoplasm or the nucleus,
and they may be bound to co-repressors in the absence of the ligand (Olefsky, 2001).
The DBD binds to specific Hormone Response Element (HRE) sequences that exist as
half-sites separated by variable length nucleotide repeats. The receptors can exist as
homo or heterodimers with each partner binding to specific HRE sequences. The DBD
consists of two zinc fingers that set the NHR apart from all other DNA binding proteins
(Mangelsdorf et al., 1995). The N-terminal and C-terminal domains are variable, as is the
hinge region between the LBD and DBD. The transcriptional activity of the hormone
receptor is mediated through Activation Function 1 and Activation Function 2 domains
(AF-1 and AF-2) present on the C- and N- termini respectively. Ligand binding to the
NHR-LBD results in a conformational change and leads to the activation of downstream
transcriptional pathways. Several other molecules such as co-activators, co-repressors,
co-integrators, chromatin remodeling proteins, and histone deacetylation proteins are
recruited at the site of DNA binding in order to facilitate or inhibit the transcription of the
target DNA into mRNA. These proteins regulate the interaction of the NHRs with their
target genes involved in development, cell differentiation and organ physiology
(Mangelsdorf et al., 1995). The following section addresses a specific type of NHR - the
Estrogen Receptor (ER) - and its role in cellular function.
Estrogen Receptor
The estrogen receptor (ER), a member of the nuclear receptor superfamily, is an
estrogen activated transcription factor that binds to short DNA sequences called
estrogen response elements (EREs) located in the vicinity of estrogen regulated genes
(Parker, 1991). There are two known isoforms of the ER, ERa and ERp, and these
proteins are encoded by separate genes. The ERa isoform (66 KDa) was the first to be
cloned in the 1980s from MCF-7 cells (Green et al., 1986; Greene et al., 1986; Walter et
al., 1985). The P isoform (54 KDa) was cloned in the 1990s from rat prostate and ovary
(Kuiper et al., 1996). The distribution of these isoforms varies from tissue to tissue, and
each isoform may also localize to specific cellular subtypes within a tissue. Several
studies show that ERa mRNA is predominant in the uterus, liver, mammary gland, testis,
pituitary, kidney, heart and skeletal muscle, whereas ERp transcripts are significantly
expressed in the ovary and the prostate (Bardin et al., 2004; Brandenberger et al., 1998;
Brandenberger et al., 1999; Couse et al., 1997; Lazennec, 2006). The relative level of
the two isoforms in a particular tissue type likely determines the impact of estrogen in
that tissue, because the two ERs differ in their functional response to estrogen, as
described below.
ERa and ERp have similar structural modularity (Figure 1.2B, adapted from Ogawa et al.,
1998), with a highly conserved DBD and a moderately conserved LBD. However, there
are significant differences in other domains that are expected to affect the transcriptional
activity of the receptors. Figure 1.2B shows the structural aspects of the two isoforms,
and the percentage identity between ERa and ERp is also denoted. The A/B domain
shows 30% identity, and also contains the N-terminal AF-1, a constitutive contributor to
the transcriptional activity of the ERs. The activation domains within the two isoforms
are not functionally equivalent - ERa displays AF-1 activity, whereas the ERP AF-1 is
shown to be functionally inactive (Hall and McDonnell, 1999; Kuiper et al., 1998a; Kuiper
et al., 1998b).
The hinge region (D domain), which contains the first Nuclear
Localization Signal (NLS) is not well conserved. Lastly, the E/F region displays 53%
homology, and contains the LBD, a coregulator binding surface, a dimerization domain,
a second NLS and AF-2, a ligand-dependent activation function (Pearce and Jordan,
2004).
In spite of the 53% homology in the LBD, differences in hormone action arise due to
differences in hormone-LBD interactions. ERa and ERP demonstrate similar binding
affinities for estradiol (KD of 0.05 nM for ERa and 0.07 nM for ERP), but their binding
affinities for other agonists, partial agonists and antagonists show subtle differences
(Kuiper et al., 1997; Kuiper et al., 1998a). Variations in these interactions likely result in
functional consequences for ERa and ERp. Although the DBDs of the isoforms are
highly conserved, it has been observed that ERa has a higher affinity for DNA than ERp.
This has been postulated to be due to the differences in the ability of the isoforms to
dimerize (Cowley et al., 1997; Pettersson et al., 1997). The isoforms have been shown
to interact with DNA as dimers; depending on the relative levels of the isoforms in cells,
the receptor can form ERa homodimers, ERP homodimers, or ERa-ERp heterodimers.
Depending on which one of the dimers is formed, there can exist three potential
alternative pathways of estrogen signaling (Pettersson et al., 1997). It is likely that there
are target genes that interact preferentially with either of the homodimers or the
heterodimer, and this would implicate a larger regulatory potential for the ligand. This
would also imply a mechanism by which the relative isoform levels modulate the target
gene activity in tissues. The distinct transcriptional profiles of the two isoforms likely
dictate the differential responses to agonists and antagonists in target cells and tissues.
In the absence of the hormone, ER is associated with the nuclear compartment of the
cell (Htun et al., 1999; King and Greene, 1984), indicating that the receptor contains an
NLS that is constitutively active independent of ligand binding. In addition, reports from
the laboratories of Stenoien and Htun have shown that there is a dramatic change in the
pattern of nuclear distribution of the ER from reticular when unliganded to punctuate in
the presence of either an agonist or an antagonist (Htun et al., 1999; Stenoien et al.,
2000; Stenoien et al., 2001). However, recent evidence suggests that a portion of the
ER population might also localize to the plasma membrane where it regulates protein
kinase cascades in response to estrogen (Razandi et al., 2004; Xu et al., 2004). The
supposed interactions between the plasma membrane bound liganded ERs and
membrane-associated molecules such as ion channels, G-proteins, and growth factor
29
receptors lead to the activation of downstream cascades such as MAP Kinase and P13
Kinase (Fu and Simoncini, 2008).
Unliganded ER in the nucleus is maintained in a high affinity hormone binding
conformation by the Heat Shock Protein 90 (HSP90) chaperone complex (Fliss et al.,
2000; Oxelmark et al., 2003; Segnitz and Gehring, 1995; Segnitz and Gehring, 1997).
Upon ligand binding, the classical ER response pathway is activated - dissociation of
HSPs from the ER, receptor dimerization and association with specific EREs to
modulate transcription initiation from nearby promoters. EREs in several genes have
been identified and most of them conform to a core consensus sequence, a thirteen
nucleotide segment with ten nucleotides forming an inverted repeat (Driscoll et al., 1998).
The
perfect
ERE
was
determined
to
have
the
consensus
sequence
5'-GGTCANNNTGACC-3', where N represents any nucleotide (Driscoll et al., 1998;
Klein-Hitpass et al., 1988). However, many other EREs with one or two deviations from
the core sequence still displayed ER binding provided there were appropriate flanking
sequences, as demonstrated by Driscoll et al. (1998).
In addition to responding via the classical pathway, the ER can also act via by proteinprotein tethering interactions at AP-1 and Sp1 sites (Kushner et al., 2000b; Kushner et
al., 2000a; Saville et al., 2000; Webb et al., 1999), which would obviate the need for
strict adherence to the consensus ERE sequence. In cells transfected with ERa or ERP
and an AP-1 or Sp1 reporter, ligand stimulation induced reporter activity to different
extents, indicating that this non-classical pathway does get activated by estrogens and
antiestrogens (Saville et al., 2000; Webb et al., 1995). ER also gets phosphorylated at
various sites in AF1 and DBD by kinases and ligands, inducing a downstream cascade
of growth regulatory pathways (Ali et al., 1993; Lahooti et al., 1994; Le et al., 1994). It is
clear that cell specific regulation of the estrogenic response is achieved through various
means - the ratio of ERa to ERp, pathways activated (classical or non-classical), ligandreceptor affinity and ER phosphorylation.
The genes activated through estrogen response pathways include progesterone
receptor (PR) (Osborne et al., 1980), pS2 (Jakowlew et al., 1984; Masiakowski et al.,
1982), the early response proto-oncogene c-myc (Schuchard et al., 1993), cathepsin D
(Morisset et al., 1986; Rochefort, 1990), HSP 27 (Moretti-Rojas et al., 1988), aldolase A,
a-tubulin and glyceraldehyde-3-phosphate (May and Westley, 1988). Some of the above
genes contain the classical palindromic ERE sequences upstream of their promoter
region, whereas some others contain non-consensus ERE half-sites and GC-rich motifs,
which might be sites where the non-classical activation of ER dominates (Pearce and
Jordan, 2004). In addition to these genes, a number of estrogen-induced mRNAs have
been identified whose function is yet unknown (May and Westley, 1995).
ER and Ovarian Cancer
Ovaries are the site of estrogen biosynthesis, and they are the main target tissue of
estrogen action. Estrogens are essential to the healthy functioning of the female
reproductive system. In addition to their functions related to reproductive activity,
estrogens are also necessary for the differentiation and proliferation of healthy breast
epithelium (Russo and Russo, 1998). The benefits of estrogen in the cardiovascular
system and in promoting bone health have been well-documented (Huang and Kaley,
2004). In fact, estrogen deficiency has been strongly correlated with age-dependent
bone loss in both sexes (Riggs et al., 2002). Estrogens have also been shown to
possess neurotrophic and neuroprotective effects, in additional to influencing memory
mechanisms, cognition, and fine motor skills (Maggi et al., 2004; Morissette et al., 2008).
The predominant intra-cellular estrogen is 17p-estradiol. Estradiol is mainly produced
from follicular thecal cells and regulates several functional aspects of ovarian cells. ERa
and ERp - the mediators of estrogen action - are expressed in normal OSE cells as well
as in malignant cells. Although the etiological origins of EOC have not been fully
identified, several studies point to the role of estrogen in promoting tumorigenesis.
Estrogen has been shown to induce proliferation in many ER(+) cancer cells, and
antiestrogens have been shown to inhibit the mitogenic effect of estrogen in those cells
(Langdon et al., 1994; Nash et al., 1989). However, there appears to be a noteworthy
variation in estrogen-mediated gene expression between ER(+) breast cancer and
ovarian cancer cells. For instance, pS2 is not estrogen regulated in several ER(+)
ovarian cancer cells, unlike in breast cancer cell lines. HER2, which is downregulated by
estrogen in breast cancer cells, does not seem to respond similarly in ovarian cancer
cells (Rochefort, 1998; Rochefort, 1999; Schaner et al., 2003).
The study of the levels of effectors of estrogen action - ERa and ERp - is crucial in order
to understand how estrogen regulation is carried out in ovarian cancer cells. The levels
of the receptor isoforms in normal and malignant ovaries have been studied extensively,
but many of the studies yield conflicting results (Lau et al., 1999; Reviewed in Pearce
and Jordan, 2004). However, a majority of the studies show that the ERa/ERp ratio has
been upregulated in ovarian cancer compared to the normal OSE cells (Cunat et al.,
2004; Pujol et al., 1998). This interesting result supports a scenario where ERa becomes
the dominant player in ovarian cancer, and ERp plays a protective role presumably by
decreasing growth stimulation of cells or by inhibiting ERa mediated proliferation. It is
32
possible that cells that are ERa dominant have a selective growth advantage and
proceed towards malignancy. Consequently, in the context of therapy, the isoforms will
have to be targeted using different approaches. My dissertation is focused on probing
the mechanism of novel antitumor agents that have been developed in the Essigmann
laboratory to target the ER.
Since ERx expression seems to be dominant in EOC
(compared to ERP), the mechanistic studies described subsequently in this dissertation
are focused on the ERa isoform. Henceforth, any references to the ER will implicitly
mean ERa, unless specified otherwise.
Rational Design of Novel Genotoxicants
that Target ER(+) Tumors
There is evidence from numerous studies to suggest that EOC growth could actually be
estrogen sensitive, and this finding holds much promise for ER as a potential target for
therapeutic development (Cunat et al., 2004; Ho, 2003; Masood et al., 1989; Nash et al.,
1989; Rose et al., 1990). We have synthesized novel genotoxicants that leverage the
presence -of ER in tumor cells to- subject selectively ER(+) cells to the consequences of
interacting with the bifunctional antitumor agents (Mitra et al., 2002; Rink et al., 1996).
The intellectual origins of this synthesis have been based on the lessons learned from
studying the mechanisms of action of drugs such as cisplatin (Figure 1.1) in order to
develop better, more selective anticancer agents. One of the cisplatin mechanisms
suggests that the combination of a DNA damaging functionality with a ligand for a tumorspecific protein could provide a novel way to improve the therapeutic index of antitumor
agents. The hypothesized mechanisms of action of this class of drugs are shown in
Figure 1.3. Research has shown that DNA lesions formed in cells by certain carcinogens
and drugs have the capability to attract transcription factors, often displaying very tight
binding (Donahue et al., 1990; MacLeod et al., 1995). Such tight binding can potentially
give rise to one or more of the two scenarios: (i) the interaction inhibits access of DNA
repair proteins to the lesion, leading to adduct persistence and toxicity (Brown et al.,
1993; McA'Nulty et al., 1996; McA'Nulty and Lippard, 1996; Treiber et al., 1994), or (ii)
the interaction leads to decreased cellular access to the transcription factors
sequestered by the adduct (hijacking hypothesis, explained in Figure 1.3), which can
compromise cellular processes, eventually leading to cell death (Treiber et al., 1994).
The genotoxicants synthesized in the Essigmann lab contain a DNA-alkylating aniline
mustard moiety that is tethered to a ligand for the ER. The mechanism of action of this
class of agents is shown in Figure 1.4. The aniline mustard moiety is based on FDA
approved drugs such as chlorambucil and melphalan (Figure 1.1). The linker that
connects the steroid moiety to the nitrogen mustard has been developed so as to permit
the DNA adducts formed by the mustard moiety to present the steroid domain in such a
way to enable efficient interaction with the ER. The drug candidates have also been
optimized for ER binding, enhanced solubility and stability against esterases. The
structural and the design aspects of the two compounds are described in the following
sections.
E2-7a-C6NC2-Mustard
E2-7a combines a DNA damaging aniline mustard moiety with an estradiol moiety that
serves as a ligand for the ER, as shown in Figure 1.4. The synthesis of the drug is
described in detail in Mitra et al. (2002). The attachment between the two moieties was
34
made at the 7a position of estradiol. This choice was based on reports that large alkyl
groups could be attached to the 7a position without loss of ER affinity (Bowler et al.,
1989; Bucourt et al., 1978; DaSilva and van Lier, 1990). I shall describe the background
studies that have been done with E2-7a in Chapter 2, in order to put in perspective my
studies with the toxicant in an ovarian cancer background. In the next two chapters, I
shall report some of the very interesting findings we observed with our study on E2-7a in
an ovarian cancer background.
Phenylindole-C6NC2-Mustard
Chronologically, this compound is first of the two programmable genotoxicants that was
developed in the Essigmann lab (Rink et al., 1996). The drug consists of a
2-phenylindole (2PI) moiety that serves as a ligand for the ER as shown in Figure 1.4.
Derivatives of 2PI have been shown to interact well with the ER in competition assays
with the natural ligand estradiol (von Angerer et al., 1984). The 2PI moiety in this
compound is linked to an aniline mustard whose chemistry and ability to alkylate DNA
are well understood (Lawley, 1966). The synthesis and preliminary toxicological studies
of this drug have been described in the publication by Rink et al. (1996). My studies on
this compound are in the ovarian cancer scenario, and the experiments described in this
dissertation that focus on the 2PI compounds have been done in collaboration with Dr.
Pei-Sze Ng (Essigmann laboratory).
E2-7a and 2PI compounds have been discussed in separate chapters because they
seem to differ in their mechanism of action - Chapters 2 and 3 will focus on my work on
E2-7a, its biological consequences followed by mechanistic understanding of the effects
of the genotoxicant. Chapters 4 will focus on the study of the phenylindole compound.
For each of the drugs, I shall discuss effects on cultured cells (ability to inhibit cell growth,
biological pathways activated and DNA adduct formation), discuss whether the
mechanism of action of the compounds aligns with our hypothesis, and assess their
potential as chemotherapeutic agents. I shall predominantly focus on the work that I
have done, but in order to make a complete story, I will occasionally discuss the work
done by my colleagues and reference accordingly.
-Chapter 1-
Structures of relevant chemotherapeutic agents
CL
,,NH
3
CPtN.
Cisplatin
C1
NH3
H 3 C-N~
Mechlorethamine
I
Melphalan
Chlorambucil
0
HO"
'
Figure 1.1: Structures of chemotherapeutic agents of
interest
Structural organization of Nuclear Hormone
Receptors
C
A/B
D
E/F
LBD
DBD Hinge
Figure 1.2A: Structure and functional domains of Nuclear
Hormone Receptors. A through F are the functional
domains. The C domain isthe DBD, D is the hinge region,
and E/F contains the LBD.
Structure/ Function modularity of the ER
isoforms
C
A/B
ERax
595aa
302
263
180
1
D
ERp
E/F
-I
1
149
214 248
530aa
Figure 1.2B: Structure and functional domains of ERa
(595 aa) and ERp (530aa). A through F are the functional
domains of the ER. The numbers inthe figure refer to the
amino acid numbers. The percentages refer to the
percentage identity between Functions AF1 and AF2
sequences are inthe A/B domain and E/F domain
respectively. The C domain is DNA Binding Domain, D is
the hinge region, and E/F contains the LBD.
-Chapter 1-
Possible mechanisms of action of
bifunctional toxicants
e
......
=s..
e.......e..eem...s.a...s.e.......esse........e......es...es...e.e.g...a
e40......
e.........e......em.a...se..
ee....
Nuclear
A. Undamaged Cell
DNA RepairG?
Enzyme
Enzyme
Protein
Promoteri
enome
11111
B.Adduct Shielc ed
From Repair
DNA Repair
. m.......
.
E[4*
Expression
of Essential Gene
4c,.C
Adduct Persists
C.Adduct Hijacl
TranscriptiornFactor
ICellD eath
U
1111
...- Diminished_
Expression
Figure 1.3: DNA adducts act as decoy binding sites for
nuclear proteins. Possible cellular outcomes are
outlined.
A. Cell with nuclear protein interacting with DNA
B. Repair Shielding - Drug-DNA adducts (lollipop) attract
the nuclear protein and are shielded from DNA repair
enzymes, leading to adduct persistence and cell death.
C. Hijacking Model - The adducts titrate the nuclear
protein away from its normal site of binding, resulting in
reduced expression of a critical gene.
-Chapter 1-
A novel agent designed to inhibit repair
processes in a cancer cell
Protein
Recognition
Domain
DNA
Damaging
Moie
Drug-DNA
Adduct
Repair
Enzyme
NORMAL CELL
CANCER CELL
ER
Adduct is repaired by repair
enzyme and is rendered nontoxic
Adduct is "shielded" by ER,
and the complex is toxic to
cell
Figure 1.4: The compounds we designed consist of a
protein recognition domain linked to a DNA warhead (top
left). This compound can covalently modify DNA within a
cell (top right). In non-cancerous cells, the DNA adduct is
removed by the repair machinery (lower left). Incancerous
cells, a tumor specific protein is aberrantly expressed, as is
the case with the ER inmany breast cancers. An
interaction between the ER and the protein recognition
domain of the designed molecules could inhibit the repair
of the adduct, render the complex toxic to the cancer cell
and cause cell death (lower right).
-Chapter 1-
Structures of bifunctional genotoxicants
that target the ER
2PI-C6NC2-mustard
HO
HN
HNHO
A.C1
0
E2-7a -C6NC2-mustard
Figure 1.5: Structures of 2PI (top) and E2-7a (bottom).The
bifunctional toxicants have a steroid moiety that serves as
a ligand for the ER linked to an aromatic mustard. The
aromatic mustard iscapable of DNA alkylation. The linker
has a carbamate group that provides a relatively rigid
connection between the moieties and makes the
compound resistant to hydrolytic enzymes. The secondary
amino group inthe linker increases water solubility.
Reference List
1.
Ali S, Metzger D, Bornert JM, Chambon P (1993) Modulation of transcriptional
activation by ligand-dependent phosphorylation of the human oestrogen receptor
A/B region. EMBO J 12:1153-1160
2.
American Cancer Society (2008) Cancer Facts and Figures.
3.
Arai M, Utsunomiya J, Miki Y (2004) Familial breast and ovarian cancers. Int J Clin
Oncol 9:270-282
4.
Argento M, Hoffman P, Gauchez AS (2008) Ovarian cancer detection and
treatment: current situation and future prospects. Anticancer Res 28:3135-3138
5.
Armitage P, Doll (1954) The age distribution of cancer and a multi-stage theory of
carcinogenesis. Br J Cancer 8:1-12
6.
Bandera CA, Ye B, Mok SC (2003) New technologies for the identification of
markers for early detection of ovarian cancer. Curr Opin Obstet Gynecol 15:51-55
7.
Bardin A, Hoffmann P, Boulle N, Katsaros D, Vignon F, Pujol P, Lazennec G (2004)
Involvement of estrogen receptor beta in ovarian carcinogenesis. Cancer Res
64:5861-5869
8.
Bast RC, Jr., Klug TL, St JE, Jenison E, Niloff JM, Lazarus H, Berkowitz RS,
Leavitt T, Griffiths CT, Parker L, Zurawski VR, Jr., Knapp RC (1983) A
radioimmunoassay using a monoclonal antibody to monitor the course of epithelial
ovarian cancer. N EngI J Med 309:883-887
9.
Bishop JM (1991) Molecular themes in oncogenesis. Cell 64:235-248
10.
Bonadonna G, Brusamolino E, Valagussa P, Rossi A, Brugnatelli L, Brambilla C,
De LM, Tancini G, Bajetta E, Musumeci R, Veronesi U (1976) Combination
chemotherapy as an adjuvant treatment in operable breast cancer. N Engl J Med
294:405-410
11.
Borini A, Rebellato E (2008) Focus on breast and ovarian cancer. Placenta 29
Suppl B:184-190
12.
Bowler J, Lilley TJ, Pittam JD, Wakeling AE (1989) Novel steroidal pure
antiestrogens. Steroids 54:71-99
13.
Brandenberger AW, Lebovic DI, Tee MK, Ryan IP,Tseng JF, Jaffe RB, Taylor RN
(1999) Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial
and endometriosis-derived stromal cells. Mol Hum Reprod 5:651-655
14.
Brandenberger AW, Tee MK, Jaffe RB (1998) Estrogen receptor alpha (ER-alpha)
and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma
and ovarian cancer cell lines: down-regulation of ER-beta in neoplastic tissues. J
Clin Endocrinol Metab 83:1025-1028
15.
Brown SJ, Kellett PJ, Lippard SJ (1993) lxrl, a yeast protein that binds to
platinated DNA and confers sensitivity to cisplatin. Science 261:603-605
16.
Bucourt R, Vignau M, Torelli V (1978) New biospecific adsorbents for the
purification of estradiol receptor. J Biol Chem 253:8221-8228
17.
Burger RA (2007) Experience with bevacizumab in the management of epithelial
ovarian cancer. J Clin Oncol 25:2902-2908
18.
Chabner BA, Longo DL (2009) Cancer Chemotherapy and Biotherapy
19.
Chabner BA, Roberts TG, Jr. (2005) Timeline: Chemotherapy and the war on
cancer. Nat Rev Cancer 5:65-72
20. Chatterjee M, Mohapatra S, lonan A, Bawa G, li-Fehmi R, Wang X, Nowak J,Ye B,
Nahhas FA, Lu K, Witkin SS, Fishman D, Munkarah A, Morris R, Levin NK, Shirley
NN, Tromp G,Abrams J, Draghici S, Tainsky MA (2006) Diagnostic markers of
ovarian cancer by high-throughput antigen cloning and detection on arrays. Cancer
Res 66:1181-1190
21.
Clark (2006) Molecular targeted drugs and growth factor receptor inhibitors. In:
Chabner BA, Longo DL (eds)
22. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS (1997) Tissue
distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and
estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and
ERalpha-knockout mouse. Endocrinology 138:4613-4621
23. Cowley SM, Hoare S, Mosselman S, Parker MG (1997) Estrogen receptors alpha
and beta form heterodimers on DNA. J Biol Chem 272:19858-19862
24. Cunat S, Hoffmann P, Pujol P (2004) Estrogens and epithelial ovarian cancer.
Gynecol Oncol 94:25-32
25.
DaSilva JN, van Lier JE (1990) Synthesis and structure-affinity of a series of 7
alpha-undecylestradiol derivatives: a potential vector for therapy and imaging of
estrogen-receptor-positive cancers. J Med Chem 33:430-434
26. DeVita VT, Hellman S, Rosenberg SA (1997) Princples and Practice of Oncology,
5 edn
27. Donahue BA, Augot M, Bellon SF, Treiber DK, Toney JH, Lippard SJ, Essigmann
JM (1990) Characterization of a DNA damage-recognition protein from mammalian
cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the
anticancer drug cisplatin. Biochemistry 29:5872-5880
28.
Driscoll MD, Sathya G,Muyan M, Klinge CM, Hilf R, Bambara RA (1998)
Sequence requirements for estrogen receptor binding to estrogen response
elements. J Biol Chem 273:29321-29330
29.
Fliss AE, Benzeno S, Rao J, Caplan AJ (2000) Control of estrogen receptor ligand
binding by Hsp90. J Steroid Biochem Mol Biol 72:223-230
30.
Foulds (1954) The experimental study of tumor progression: a review. Cancer Res
14:327-339
31.
Fu XD, Simoncini T (2008) Extra-nuclear signaling of estrogen receptors. IUBMB
Life 60:502-510
32. Green S, Walter P, Greene G, Krust A, Goffin C, Jensen E, Scrace G,Waterfield M,
Chambon P (1986) Cloning of the human oestrogen receptor cDNA. J Steroid
Biochem 24:77-83
33. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J (1986) Sequence and
expression of human estrogen receptor complementary DNA. Science 231:11501154
34. Greenlee RT, Murray T, Bolden S, Wingo PA (2000) Cancer statistics, 2000. CA
Cancer J Clin 50:7-33
35. Hall JM, McDonnell DP (1999) The estrogen receptor beta-isoform (ERbeta) of the
human estrogen receptor modulates ERalpha transcriptional activity and is a key
regulator of the cellular response to estrogens and antiestrogens. Endocrinology
140:5566-5578
36. Hanahan D,Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70
37. Ho SM (2003) Estrogen, progesterone and epithelial ovarian cancer. Reprod Biol
Endocrinol 1:73
38. Htun H, Holth LT, Walker D, Davie JR, Hager GL (1999) Direct visualization of the
human estrogen receptor alpha reveals a role for ligand in the nuclear distribution
of the receptor. Mol Biol Cell 10:471-486
39. Huang A, Kaley G (2004) Gender-specific regulation of cardiovascular function:
estrogen as key player. Microcirculation 11:9-38
40. Hurley LH (2002) DNA and its associated processes as targets for cancer therapy.
Nat Rev Cancer 2:188-200
41.
Jacobs I, Bast RC, Jr. (1989) The CA 125 tumour-associated antigen: a review of
the literature. Hum Reprod 4:1-12
42.
Jaffe N, Frei E, Ill, Traggis D, Bishop Y (1974) Adjuvant methotrexate and
citrovorum-factor treatment of osteogenic sarcoma. N Engl J Med 291:994-997
43.
Jakowlew SB, Breathnach R, Jeltsch JM, Masiakowski P, Chambon P (1984)
Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell
line MCF-7. Nucleic Acids Res 12:2861-2878
44.
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer
statistics, 2008. CA Cancer J Clin 58:71-96
45.
King WJ, Greene GL (1984) Monoclonal antibodies localize oestrogen receptor in
the nuclei of target cells. Nature 307:745-747
46.
Klein-Hitpass L, Ryffel GU, Heitlinger E, Cato AC (1988) A 13 bp palindrome is a
functional estrogen responsive element and interacts specifically with estrogen
receptor. Nucleic Acids Res 16:647-663
47.
Krasner C (2007) Aromatase inhibitors in gynecologic cancers. J Steroid Biochem
Mol Biol 106:76-80
48.
Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson
JA (1997) Comparison of the ligand binding specificity and transcript tissue
distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870
49.
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996) Cloning of
a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A
93:5925-5930
50. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van
der BB, Gustafsson JA (1998a) Interaction of estrogenic chemicals and
phytoestrogens with estrogen receptor beta. Endocrinology 139:4252-4263
51.
Kuiper GG, Shughrue PJ, Merchenthaler 1,Gustafsson JA (1998b) The estrogen
receptor beta subtype: a novel mediator of estrogen action in neuroendocrine
systems. Front Neuroendocrinol 19:253-286
52. Kushner PJ, Agard D, Feng WJ, Lopez G, Schiau A, Uht R,Webb P, Greene G
(2000a) Oestrogen receptor function at classical and alternative response
elements. Novartis Found Symp 230:20-26
53. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P
(2000b) Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311317
54. Lahooti H,White R, Danielian PS, Parker MG (1994) Characterization of liganddependent phosphorylation of the estrogen receptor. Mol Endocrinol 8:182-188
55.
Langdon SP, Hirst GL, Miller EP, Hawkins RA, Tesdale AL, Smyth JF, Miller WR
(1994) The regulation of growth and protein expression by estrogen in vitro: a
study of 8 human ovarian carcinoma cell lines. J Steroid Biochem Mol Biol 50:131135
56. Langdon SP, Ritchie A, Young K, Crew AJ, Sweeting V, Bramley T, Hillier S,
Hawkins RA, Tesdale AL, Smyth JF, . (1993) Contrasting effects of 17 betaestradiol on the growth of human ovarian carcinoma cells in vitro and in vivo. Int J
Cancer 55:459-464
57. Lau KM, Mok SC, Ho SM (1999) Expression of human estrogen receptor-alpha
and -beta, progesterone receptor, and androgen receptor mRNA in normal and
malignant ovarian epithelial cells. Proc Natl Acad Sci U S A 96:5722-5727
58.
Lawley PD (1966) Effects of some chemical mutagens and carcinogens on nucleic
acids. Prog Nucleic Acid Res Mol Biol 5:89-131
59.
Lazennec G (2006) Estrogen receptor beta, a possible tumor suppressor involved
in ovarian carcinogenesis. Cancer Lett 231:151-157
60.
Le GP, Montano MM, Schodin DJ, Katzenellenbogen BS (1994) Phosphorylation of
the human estrogen receptor. Identification of hormone-regulated sites and
examination of their influence on transcriptional activity. J Biol Chem 269:44584466
61.
Li M, HERTZ R,BERGENSTAL DM (1958) Therapy of choriocarcinoma and
related trophoblastic tumors with folic acid and purine antagonists. N Engl J Med
259:66-74
62. Lindgren PR, Cajander S, Backstrom T, Gustafsson JA, Makela S, Olofsson JI
(2004) Estrogen and progesterone receptors in ovarian epithelial tumors. Mol Cell
Endocrinol 221:97-104
63. Lindor NM, Petersen GM, Hadley DW, Kinney AY, Miesfeldt S, Lu KH, Lynch P,
Burke W, Press N (2006) Recommendations for the care of individuals with an
inherited predisposition to Lynch syndrome: a systematic review. JAMA 296:15071517
~jI
64.
Lukanova A, Kaaks R (2005) Endogenous hormones and ovarian cancer:
epidemiology and current hypotheses. Cancer Epidemiol Biomarkers Prev 14:98107
65. MacLeod MC, Powell KL, Tran N (1995) Binding of the transcription factor, Spl, to
non-target sites in DNA modified by benzo[a]pyrene diol epoxide. Carcinogenesis
16:975-983
66. Maggi A, Ciana P, Belcredito S, Vegeto E (2004) Estrogens in the nervous system:
mechanisms and nonreproductive functions. Annu Rev Physiol 66:291-313
67. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K,
Blumberg B, Kastner P, Mark M, Chambon P, Evans RM (1995) The nuclear
receptor superfamily: the second decade. Cell 83:835-839
68. Masiakowski P, Breathnach R, Bloch J,Gannon F, Krust A, Chambon P (1982)
Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human
breast cancer cell line. Nucleic Acids Res 10:7895-7903
69.
Masood S, Heitmann J, Nuss RC, Benrubi GI (1989) Clinical correlation of
hormone receptor status in epithelial ovarian cancer. Gynecol Oncol 34:57-60
70.
May FE, Westley BR (1988) Identification and characterization of estrogenregulated RNAs in human breast cancer cells. J Biol Chem 263:12901-12908
71.
May FE, Westley BR (1995) Estrogen regulated messenger RNAs in human breast
cancer cells. Biomed Pharmacother 49:400-414
72.
McA'Nulty MM, Lippard SJ (1996) The HMG-domain protein lxrl blocks excision
repair of cisplatin-DNA adducts in yeast. Mutat Res 362:75-86
73.
McA'Nulty MM, Whitehead JP, Lippard SJ (1996) Binding of Ixrl, a yeast HMGdomain protein, to cisplatin-DNA adducts in vitro and in vivo. Biochemistry
35:6089-6099
74.
Merajver SD, Pham TM, Caduff RF, Chen M, Poy EL, Cooney KA, Weber BL,
Collins FS, Johnston C, Frank TS (1995) Somatic mutations in the BRCA1 gene in
sporadic ovarian tumours. Nat Genet 9:439-443
75.
Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu
Q, Cochran C, Bennett LM, Ding W, . (1994) A strong candidate for the breast and
ovarian cancer susceptibility gene BRCA1. Science 266:66-71
76.
Mitra K, Marquis JC, Hillier SM, Rye PT, Zayas B, Lee AS, Essigmann JM, Croy
RG (2002) A rationally designed genotoxin that selectively destroys estrogen
receptor-positive breast cancer cells. J Am Chem Soc 124:1862-1863
77.
Moretti-Rojas I, Fuqua SA, Montgomery RA, 111,
McGuire WL (1988) A cDNA for
the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells. Breast
Cancer Res Treat 11:155-163
78.
Morisset M, Capony F, Rochefort H (1986) Processing and estrogen regulation of
the 52-kilodalton protein inside MCF7 breast cancer cells. Endocrinology
119:2773-2782
79. Nash JD, Ozols RF, Smyth JF, Hamilton TC (1989) Estrogen and anti-estrogen
effects on the growth of human epithelial ovarian cancer in vitro. Obstet Gynecol
73:1009-1016
80.
Noy N (2007) Ligand specificity of nuclear hormone receptors: sifting through
promiscuity. Biochemistry 46:13461-13467
81.
O'Donnell AJ, Macleod KG, Burns DJ, Smyth JF, Langdon SP (2005) Estrogen
receptor-alpha mediates gene expression changes and growth response in ovarian
cancer cells exposed to estrogen. Endocr Relat Cancer 12:851-866
82. Olefsky JM (2001) Nuclear receptor minireview series. J Biol Chem 276:3686336864
83. Osborne CK, Yochmowitz MG, Knight WA, Ill, McGuire WL (1980) The value of
estrogen and progesterone receptors in the treatment of breast cancer. Cancer
46:2884-2888
84. Oxelmark E, Knoblauch R, Arnal S, Su LF, Schapira M, Garabedian MJ (2003)
Genetic dissection of p23, an Hsp90 cochaperone, reveals a distinct surface
involved in estrogen receptor signaling. J Biol Chem 278:36547-36555
85. Ozols RF (2005) Treatment goals in ovarian cancer. Int J Gynecol Cancer 15
Suppl 1:3-11
86. Pal T, Permuth-Wey J, Betts JA, Krischer JP, Fiorica J, Arango H, LaPolla J,
Hoffman M, Martino MA, Wakeley K,Wilbanks G, Nicosia S, Cantor A, Sutphen R
(2005) BRCA1 and BRCA2 mutations account for a large proportion of ovarian
carcinoma cases. Cancer 104:2807-2816
52
87.
Papac RJ (2001) Origins of cancer therapy. Yale J Biol Med 74:391-398
88.
Parker MG (1991) Nuclear Hormone Receptors
89.
Parker, Jensen EV (1991) Overview of the Nuclear Receptor Family.
90.
Pearce ST, Jordan VC (2004) The biological role of estrogen receptors alpha and
beta in cancer. Crit Rev Oncol Hematol 50:3-22
91.
Pettersson K, Grandien K, Kuiper GG, Gustafsson JA (1997) Mouse estrogen
receptor beta forms estrogen response element-binding heterodimers with
estrogen receptor alpha. Mol Endocrinol 11:1486-1496
92. Pujol P, Rey JM, Nirde P, Roger P, Gastaldi M, Laffargue F, Rochefort H,
Maudelonde T (1998) Differential expression of estrogen receptor-alpha and -beta
messenger RNAs as a potential marker of ovarian carcinogenesis. Cancer Res
58:5367-5373
93. Rao GG, Miller DS (2005) Clinical applications of hormonal therapy in ovarian
cancer. Curr Treat Options Oncol 6:97-102
94. Razandi M, Pedram A, Merchenthaler I, Greene GL, Levin ER (2004) Plasma
membrane estrogen receptors exist and functions as dimers. Mol Endocrinol
18:2854-2865
95.
Renan MJ (1993) How many mutations are required for tumorigenesis?
Implications from human cancer data. Mol Carcinog 7:139-146
96.
Riggs BL, Khosla S, Melton LJ, Ill (2002) Sex steroids and the construction and
conservation of the adult skeleton. Endocr Rev 23:279-302
53
97. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2001) Risk factors for epithelial borderline ovarian tumors: results of a
Swedish case-control study. Gynecol Oncol 83:575-585
98. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2002a) Risk factors for invasive epithelial ovarian cancer: results from
a Swedish case-control study. Am J Epidemiol 156:363-373
99.
Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Weiderpass E, Persson IR (2002b) Hormone replacement therapy and the risk of
invasive epithelial ovarian cancer in Swedish women. J Natl Cancer Inst 94:497504
100. Riman T, Dickman PW, Nilsson S, Nordlinder H,Magnusson CM, Persson IR
(2004) Some life-style factors and the risk of invasive epithelial ovarian cancer in
Swedish women. Eur J Epidemiol 19:1011-1019
101. Rink SM, Yarema KJ, Solomon MS, Paige LA, Tadayoni-Rebek BM, Essigmann
JM, Croy RG (1996) Synthesis and biological activity of DNA damaging agents that
form decoy binding sites for the estrogen receptor. Proc Natl Acad Sci U S A
93:15063-15068
102. Risch HA (2002) Hormone replacement therapy and the risk of ovarian cancer.
Gynecol Oncol 86:115-117
103. Rochefort H (1990) Cathepsin D in breast cancer. Breast Cancer Res Treat 16:3-
13
104. Rochefort H (1998) [Estrogens, cathepsin D and metastasis in cancers of the
breast and ovary: invasion or proliferation?]. C R Seances Soc Biol Fil 192:241-251
105. Rochefort H (1999) [Estrogen-induced genes in breast cancer, and their medical
importance]. Bull Acad Natl Med 183:955-968
106. Rose PG, Reale FR, Longcope C, Hunter RE (1990) Prognostic significance of
estrogen and progesterone receptors in epithelial ovarian cancer. Obstet Gynecol
76:258-263
107. Russo IH, Russo J (1998) Role of hormones in mammary cancer initiation and
progression. J Mammary Gland Biol Neoplasia 3:49-61
108. Rutherford T, Brown WD, Sapi E, Aschkenazi S, Munoz A, Mor G (2000) Absence
of estrogen receptor-beta expression in metastatic ovarian cancer. Obstet Gynecol
96:417-421
109. Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA,
Safe S (2000) Ligand-, cell-, and estrogen receptor subtype (alpha/beta)dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:53795387
110. Schaner ME, Ross DT, Ciaravino G, Sorlie T, Troyanskaya 0, Diehn M, Wang YC,
Duran GE, Sikic TL, Caldeira S, Skomedal H, Tu IP, Hernandez-Boussard T,
Johnson SW, O'Dwyer PJ, Fero MJ, Kristensen GB, Borresen-Dale AL, Hastie T,
Tibshirani R, van de RM, Teng NN, Longacre TA, Botstein D, Brown PO, Sikic BI
(2003) Gene expression patterns in ovarian carcinomas. Mol Biol Cell 14:43764386
111. Schildkraut JM, Schwingl PJ, Bastos E, Evanoff A, Hughes C (1996) Epithelial
ovarian cancer risk among women with polycystic ovary syndrome. Obstet Gynecol
88:554-559
112. Schuchard M, Landers JP, Sandhu NP, Spelsberg TC (1993) Steroid hormone
regulation of nuclear proto-oncogenes. Endocr Rev 14:659-669
113. Segnitz B, Gehring U (1995) Subunit structure of the nonactivated human estrogen
receptor. Proc Natl Acad Sci U S A 92:2179-2183
114. Segnitz B, Gehring U (1997) The function of steroid hormone receptors is inhibited
by the hsp90-specific compound geldanamycin. J Biol Chem 272:18694-18701
115. Slotman BJ, Rao BR (1988) Ovarian cancer (review). Etiology, diagnosis,
prognosis, surgery, radiotherapy, chemotherapy and endocrine therapy. Anticancer
Res 8:417-434
116. Stenoien DL, Mancini MG, Patel K, Allegretto EA, Smith CL, Mancini MA (2000)
Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1.
Mol Endocrinol 14:518-534
117. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, Mancini
MA (2001) FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and
proteasome-dependent. Nat Cell Biol 3:15-23
118. Treiber DK, Zhai X, Jantzen HM, Essigmann JM (1994) Cisplatin-DNA adducts are
molecular decoys for the ribosomal RNA transcription factor hUBF (human
upstream binding factor). Proc Natl Acad Sci U S A 91:5672-5676
119. von Angerer E, Prekajac J, Strohmeier J (1984) 2-Phenylindoles. Relationship
between structure, estrogen receptor affinity, and mammary tumor inhibiting
activity in the rat. J Med Chem 27:1439-1447
120. Walker G, MacLeod K, Williams AR, Cameron DA, Smyth JF, Langdon SP (2007)
Estrogen-regulated gene expression predicts response to endocrine therapy in
patients with ovarian cancer. Gynecol Oncol 106:461-468
121. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E,
Scrace G, Waterfield M, . (1985) Cloning of the human estrogen receptor cDNA.
Proc Natl Acad Sci U S A 82:7889-7893
122. Webb P, Lopez GN, Uht RM, Kushner PJ (1995) Tamoxifen activation of the
estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9:443-456
123. Webb P, Nguyen P, Valentine C, Lopez GN, Kwok GR, McInerney E,
Katzenellenbogen BS, Enmark E, Gustafsson JA, Nilsson S, Kushner PJ (1999)
The estrogen receptor enhances AP-1 activity by two distinct mechanisms with
different requirements for receptor transactivation functions. Mol Endocrinol
13:1672-1685
124. Xu Y, Traystman RJ, Hurn PD, Wang MM (2004) Membrane restraint of estrogen
receptor alpha enhances estrogen-dependent nuclear localization and genomic
function. Mol Endocrinol 18:86-96
125. Yap TA, Carden CP, Kaye SB (2009) Beyond chemotherapy: targeted therapies in
ovarian cancer. Nat Rev Cancer 9:167-181
126. Zheng L, Annab LA, Afshari CA, Lee WH, Boyer TG (2001) BRCA1 mediates
ligand-independent transcriptional repression of the estrogen receptor. Proc Natl
Acad Sci U S A 98:9587-9592
Chapter 2
E2-7a - Biological
Consequences in Ovarian
Cancer Cells: Cytotoxicity, DNA
Damage and Cell Death
Introduction
Estradiol-C6NC2-N,N-bis-(2-chloroethyl)-aniline (E2-7a) is a programmable therapeutic
that has been designed to target estrogen receptor positive (ER(+)) tumors such as
breast and ovarian cancers. The intellectual origins of E2-7a design stem from the
mechanistic understanding of cisplatin action. Cisplatin (cis-diamminedichloroplatinum(II)
or cis-DDP) has shown spectacular efficacy in the treatment of testicular and ovarian
malignancies and offers great opportunities for probing new mechanisms of inducing
toxicity. The success of cisplatin used alone or in combination with drugs such as
paclitaxel, docetaxel, gemcitabine and doxorubicin in treating testicular tumors and
delaying the recurrence of ovarian cancers has interested researchers world-wide, and
has stimulated extensive research to understand cellular response to the drug (Boulikas
and Vougiouka, 2004). It is known that cisplatin reacts with DNA (Bruhn et al., 1990),
and induces apoptosis in treated cells. There is evidence, both from our laboratory and
researchers elsewhere, that DNA-lesions formed by cisplatin have the ability to attract
cellular proteins such as High Mobility Group (HMG) box proteins and bind them with
very high affinity (Donahue et al., 1990; Pil and Lippard, 1992). In addition, proteins such
as hMSH2 that bind to the cisplatin-DNA lesions are selectively expressed in organs in
which cisplatin is clinically effective as an anticancer agent (Wilson et al., 1995). There
are two routes by which such DNA-drug-protein complexes can exert their effect. First,
the tight binding might "shield" the adduct and prevent DNA repair enzymes from
accessing the lesion (Huang et al., 1994). The alternative mechanism arises from the
observation that the tight binding proteins are usually critical transcription factors and by
"hijacking" these factors away from their sites of action, the adduct causes reduction in
expression levels of genes essential for survival. We have tailored the idea of a dual
mode of action - 1) alkylation and 2) ability to interact with tumor-specific proteins - to
synthesize a series of alkylating compounds that also contain a tumor-specific protein
recognition moiety.
The evolution of a newer generation of drugs based on the cisplatin mechanism of action
will be invaluable in order to achieve efficacy similar to or better than cisplatin, yet
circumvent the toxic side effects and development of resistance associated with cisplatin
therapy. Cisplatin shows high efficacy in the treatment of testicular tumors, but concerns
regarding its clinically significant toxicity and its side effects such as nephrotoxicity and
central and peripheral neurotoxicity still remain (Reed, 2006). In addition, several
cancers including ovarian cancers develop resistance after the first treatment regimen of
cisplatin, and this consequently leads to very high relapse rates (Reed, 2006).
Combination therapies of platinum compounds with agents such as taxol, doxorubicin,
and cyclophosphamide have their own dose-limiting side effects as well (DeVita et al.,
1997). In an effort to overcome the above mentioned undesirable off-target effects of
cisplatin, we have extrapolated the extensive knowledge available on the drug to
synthesize a series of "re-programmed" genotoxicants that are designed to target tumors
more selectively and are therefore more efficacious than cisplatin.
E2-7a: Synthesis Strategy
Our strategy was to select a DNA damaging agent that could also attract a tumorspecific protein. This approach led us to tether an alkylating aniline mustard moiety to
the ER, which functioned as the protein recognition domain of our compound of interest
E2-7a. E2-7a was synthesized as per the steps outlined by Mitra et al. (2002). The linker
group connecting estradiol and the aniline mustard was attached at the 7a position of
estradiol based on reports that large groups could be attached to that position without
affecting binding affinity to the ER (Bowler et al., 1989; Bucourt et al., 1978; DaSilva and
van Lier, 1990). The aniline mustard N,N-bis-(2-chloroethyl)-aniline was chosen over an
alkyl nitrogen mustard such as mechlorethamine, because the aniline mustard reacts
relatively slowly (it has lower toxicity compared to the more reactive alkyl mustards) and
the DNA adducts formed by the aniline mustard are readily repaired (Essigmann et al.,
2001b; Pratt et al., 1994). Therefore, using the aniline mustard as the alkylating moiety
would potentially reduce the toxicity of the compound in non-target tissues. Sharma and
others (2004) from the Essigmann lab synthesized a series of variants of the parent E27a compound in order to fine-tune the biophysical properties of the compound. This
series of compounds contained the same alkylation moiety and estradiol moiety as did
E2-7a, but differed in their linker functional groups and lengths. These compounds were
tested for their binding affinity to the ER, their ability to covalently modify DNA and their
(differential) toxicity towards ER(+) MCF-7 and ER(-) MDA-MB-231 breast cancer cell
lines. The systematic effort to optimize linker parameters by Sharma et al. helped us
select the compound best suited for our further studies - the parent E2-7aX compound
displayed the optimal balance between solubility, DNA reactivity, ER binding and
differential toxicity between ER(+) and ER(-) cell lines. The linker portion of E2-7a
contains the carbamate and amine functional groups and is stable to hydrolytic cleavage
and enhances the solubility of the drug.
E2-7a: Background Studies
The Essigmann lab has conducted extensive research on E2-7at to characterize its
biophysical and toxicological properties. The findings from these studies have been
summarized in this section, some of which can also be found in the publication by Mitra
et al. (2002). The history of E2-7a described here leads directly to the research goals
discussed in this dissertation.
The aniline mustard moiety of E2-7a is predicted to react with DNA in vitro to alkylate
predominantly at the N7 position of guanine because of the highly nucleophilic nature of
the nitrogen atom on the guanine. To determine the identity of DNA adducts formed by
E2-7a, DNA treated with drug was subjected to reversed-phase HPLC and analyzed by
full scan electrospray ionization mass spectrometry (ESI-MS) (Mitra et al., 2002). A
prominent ion signal was observed at a mass consistent with the structure where one
arm of the mustard is attached to guanine, and the other arm contains a -OH in place of
the -Cl atom, similar to what has been reported with chlorambucil (Haapala et al., 2001).
This study provides evidence that E2-7a alkylates DNA in vitro.
The next key step in validating our hypothesis was to determine whether E2-7a can
interact with the ER, and whether interaction existed between DNA adducted to E2-7a
and the ER. The following results have also been discussed in Mitra et al. (2002).
Competitive binding studies showed that E2-7a had a favorable affinity for the rabbit
uterine ER protein, binding the receptor with 46% of the affinity of estradiol (Sharma et
al., 2004). Gel shift experiments demonstrated that E2-7a-DNA adducts are also
capable of forming a complex with ER-Ligand Binding Domain (ER-LBD). This complex
was detected by the presence of a slowly migrating band in an Electrophoretic Mobility
Shift Assay (EMSA). The binding specificity of the DNA adduct to ER-LBD was verified
by adding increasing amounts of the competitor estradiol to the DNA adduct-ER-LBD
complex. The slow migrating band corresponding to this complex disappeared, strongly
suggesting that estradiol binding to the ER might be preventing the DNA adduct from
interacting with the ER-LBD.
The cytotoxic effect of E2-7a has been studied in ER(+) and ER(-) breast cancer cell
lines (MCF-7 and MDA-MB-231 respectively), as described in Mitra et al.(2002). It was
observed that the colony forming ability was much lower in MCF-7 cells than in the ER(-)
MDA cell lines after treatment with the same dose of E2-7a. Chlorambucil was used as a
control compound, because it contains the same DNA damaging moiety as E2-7a.
Chlorambucil did not show a difference in toxicity profile between the ER(+) and ER(-)
cells. The fact that ER status affected the toxicity of E2-7a, but not that of chlorambucil,
is consistent with the role of the ER as an effector of selective toxicity of E2-7a.
E2-7a shows reasonable toxicity in vivo as well. HeLa cells engineered to express the
ER-LBD were employed to form xenografts tumors in mice. Shawn Hillier (Essigmann
laboratory) showed that E2-7a effectively inhibited the growth of ER(+) HeLa xenografts
by as much as 82%, and the treated mice showed no signs of organ specific toxicity,
indicating that the drug dose was well- tolerated (Unpublished results).
E2-7a in the Context of Ovarian Cancer
Evidence from several randomized trials has now established combination regimens of
paclitaxel with cisplatin as the front line therapy against advanced ovarian cancer
(Agarwal and Kaye, 2003; Ozols et al., 2003), but despite several efforts at optimizing
doses and durations of treatment, clinical responses remain short-lived and the
development of resistance is a huge obstacle to be overcome (Agarwal and Kaye, 2003;
Yap et al., 2009). Given the current therapy landscape in ovarian cancer, there is a
compelling need to develop more targeted approaches in order to circumvent these
issues. E2-7a seems to have tremendous potential to provide a targeted approach to
treat ER(+) ovarian tumors, potentially reducing off-target side effects as well.
This chapter focuses on the studies I have performed with E2-7a, assessing its
cytotoxicity in ovarian cancer cells and examining the cellular response to E2-7a
treatment. The biological consequences of E2-7a treatment have also been compared to
those of cisplatin treatment. Two established epithelial ovarian cancer cell lines,
OVCAR-3 (Hamilton et al., 1983) and SKOV-3 (Hua et al., 1995c) have been chosen in
order to study the cytotoxicity of the drug.
OVCAR-3 cells are derived from a progressive adenocarcinoma of the ovary and are
resistant to clinically relevant concentrations of cisplatin, adriamycin, and melphalan
(Hamilton et al., 1984). Both cultured cells and xenografts exhibit the ER (Hamilton et al.,
1983). This cell line has been chosen because it is a suitable model system of a
cisplatin-resistant ovarian cell population in which one can study the effects of alternative
therapeutics. SKOV-3 cells are epithelial cells derived from adenocarcinoma of the ovary,
and are resistant to several cytotoxic drugs including cisplatin, adriamycin and diphtheria
toxin (Morimoto et al., 1991; Morimoto et al., 1993). This cell line was specifically chosen
because the ER present in these cells retains its ligand binding ability, but seems to be
deficient in the transcriptional regulation of certain downstream growth regulatory gene
products (Hua et al., 1995b; Lau et al., 1999). As such, the cells do not respond to
estrogens such as estradiol or antiestrogens such as ICI 164,384 (Hua et al., 1995a).
This cell line serves as an appropriate model system to understand the interaction
between ER and E2-7a without involving the confounding effects of the transcriptional
activity of the receptor (these mechanistic studies will be described in detail in Chapter
3). This system helps address the mechanistic aspects of E2-7aX action and answer the
question whether ER is involved in modulating E2-7a toxicity.
OVCAR-3 and SKOV-3 cells were tested to determine their sensitivity to E2-7a and
changes in cell cycle patterns were identified in these two cell lines. In addition, some
important and interesting biomarkers of cellular responses in SKOV-3 cells were
examined and E2-7c-treated cells were probed for evidence of DNA damage. The
nitrogen mustard moiety of E2-7a is known to exert its cytotoxicity through reaction with
DNA (Sunters et al., 1992). A majority of these reaction products are monoadducts,
which result from a single alkylation event involving one of the chloroethyl arms of the
mustard reacting with DNA through the formation of an aziridinium ion intermediate
(Figure 2.1) (Balcome et al., 2004; Kohn et al., 1987; Kohn, 1996; Kundu et al., 1994).
As shown in Figure 2.1, persistence of the monoadducts could result in the formation of
interstrand or intrastrand DNA crosslinks (Hartley et al., 1991; Sunters et al., 1992), and
previous studies have shown that crosslinks are more cytotoxic than monoadducts
(O'Connor and Kohn, 1990; Sunters et al., 1992). DNA damage analysis in treated cells
will be valuable in understanding the basis of E2-7a toxicity. DNA damage triggers many
rescue/repair responses in cells, including activation of key damage signal transducers,
priming of the repair machinery and phosphorylation of checkpoint proteins. The cellular
response to the drugs E2-7a, cisplatin and chlorambucil have been examined along
these lines.
In addition to the studying the cytotoxicity in ovarian cancer cells as outlined above, I
employed a system of two cell lines (HeLa and XPA) which differ in their DNA repair
capability in order to estimate if DNA damage repair is involved in sensitizing the cells to
E2-7a. These cell lines demonstrate a significant difference in their ability to perform
Pr'-.
nucleotide excision repair (NER). HeLa cells used in this study are derived from
adenocarcinoma of the cervix and are proficient in NER (He and Ingles, 1997); these
cells were used as a control to evaluate the contribution of DNA repair to toxicity. The
XPA-deficient cells used are SV-40 transformed fibroblasts that were obtained from a
donor carrying a mutation in Xeroderma Pigmentosum Complementation Group A (XPA)
protein (Satokata et al., 1992). XPA protein is involved in excision repair of DNA and
mutation in this gene renders the cells extremely sensitive to certain kinds of DNA
damaging agents (such as UV radiation) that are dependent on NER for repair; the cells
are also sensitive to certain alkylating agents such as cisplatin and melphalan (Araujo et
al., 2001; Koberle et al., 2006; States et al., 1998). This sensitivity can be used to
evaluate whether DNA damage and its repair are involved in the toxicity towards a given
agent.
Another interesting aspect of E2-7a toxicity that is discussed in this chapter is the mode
of cell death activated by the genotoxicant in SKOV-3 cells. Cell death occurs when the
damage in the cell exceeds the threshold for its tolerance, and there are several modes
by which a cell might possibly die upon treatment with DNA damaging agents (Brown
and Attardi, 2005). The form of cell death induced by a particular agent depends on the
cell type, the type of DNA damage to which the cell is exposed, and the dose of the
agent used (Abend, 2003). Cytotoxic agents such as chlorambucil, cisplatin and
melphalan have been known to induce cell death through apoptosis, a programmed cell
death pathway (Gibb et al., 1997; Roy et al., 2000). The mode of cell death is crucial to
the action of a chemotherapeutic agent, and in several cases, resistance to apoptosis in
the tumor population leads to drug-resistant tumors (Brown and Attardi, 2005). In this
context, targeted therapies that overcome apoptosis-resistance in tumor cells by
modulating cell death pathways might be extremely effective in treating drug-resistant
tumors.
Previous studies with E2-7a in breast cancer cells have shown that these cells exhibited
typical signs of apoptosis in their response to the drug, such as caspase cleavage and
DNA fragmentation (John Marquis, Essigmann laboratory, unpublished results). We
were interested in testing whether SKOV-3 cells demonstrated a similar response to E27a; the endpoint of E2-7a treatment in ovarian cancer cells would give us handle on the
chemotherapeutic potential of the compound. The studies probing cell death pathways
activated in SKOV-3 cells are described in detail in this chapter.
Materials and Methods
Chemicals:
The bifunctional compound E2-7a used in these studies was synthesized in our
laboratory. Details of the synthetic steps and characterization of the compound have
been described in a previous publication (Mitra et al., 2002). Cisplatin (Cis), chlorambucil
(CHL), acridine orange (AO), propidium iodide (PI), ethidium bromide (EB) and
monodansylcadaverine (MDC) were obtained from Sigma-Aldrich.
Cell Culture:
SKOV-3 and NIH:OVCAR-3 (referred to as OVCAR-3) cells were obtained from the
American Type Culture Collection (ATCC, Rockville, MD). Fetal Bovine Serum (FBS)
was obtained from Thermo Fisher Scientific (Logan, UT). OVCAR-3 cells were
maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 2.5 mg/mL glucose,
2 mM GlutaMax, 1 mM sodium pyruvate and 100 mM HEPES buffer, supplemented with
0.01 mg/mL bovine insulin (Invitrogen) and 20% FBS. SKOV-3 cells were maintained in
McCoy's 5a Medium (ATCC), supplemented with 10% FBS. HeLa cells were obtained
from ATCC and XPA cells (GM04312) were from Coriell Institute for Medical Research.
HeLa and XPA cells were grown in Minimum Essential Medium (MEM) from Invitrogen
supplemented with 5 mM L-Glutamine and 10% FBS. All cell lines were grown in a
humidified 5%CO2/air atmosphere at 37*C.
For growth inhibition assays, cells were seeded in 6-well plates at 5x10 4 cells per well.
Forty eight hours later, the test compound dissolved in DMSO was added to the medium.
Cells were detached using trypLE
Express (Invitrogen) at times indicated and the
number of cells in control and drug-treated wells were determined using a Beckman
Coulter Counter. The percent growth inhibition was calculated as the ratio of cell number
in treated to that in control wells multiplied by 100. Relative sensitivities were assessed
by the concentration of compound required to inhibit growth by 50% (GI50 ; G150 is the
concentration of test drug that causes 50% growth inhibition in treated cells compared to
the untreated control cells). G150 was calculated using exponential regression analysis of
growth inhibition data.
For clonogenic assays, cells were seeded at 103 per well in 6-well plates and allowed to
attach for 24 hours. Cells were then treated with desired drug doses for 24 hours, and
the medium was replaced with fresh growth medium. Colonies were allowed to form for
4-7 days. Colonies were washed with PBS and were fixed with methanol/acetic acid and
stained with crystal violet. Colonies of 50 or more cells were counted and results were
expressed as percentage of colony formation in untreated cells.
Immunoblot Analysis:
Whole cell extracts were prepared cell lysis using RIPA buffer (Santacruz) (1XTBS, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS,
1 mM PMSF, 1 mM sodium
orthovanadate, 1 mM NaF, 50 mM Tris-HCI (pH 7.4) and protease inhibitor cocktail
(Sigma-Aldrich)). Protein concentrations were determined using Bio-Rad Bradford
reagent with Bovine Serum Albumin (BSA) standard. Equal amounts of protein were
loaded and separated on pre-cast 4-12% SDS-polyacrylamide gradient gels (Invitrogen)
in MOPS buffer system and transferred to Immobilon-P membranes (Millipore, Bedford,
MA). Membranes were blocked with 5% milk in Tris-Buffered Saline - Tween20 (0.1%
Tween20, 10 mM Tris (pH 7.4), 150 mM NaCl) and incubated with primary antibodies
overnight at 40C. The antibodies against phospho-cdc2 (rabbit polyclonal), phosphoBRCA1 (rabbit polyclonal), caspase-3 (rabbit polyclonal), caspase-9 (rabbit polyclonal)
and PARP (rabbit polyclonal) were obtained from Cell Signaling Technology (Beverly,
MA). Actin antibody (C-11,
rabbit polyclonal) was obtained from Santa Cruz
Biotechnology. Bcl-2 antibody (mouse monoclonal) was obtained from BD Pharmingen
(San Jose, CA). Anti-mouse and anti-rabbit secondary antibodies conjugated to
horseradish peroxidase were obtained from Cell Signaling Technology. Membranes
were incubated with secondary antibodies for 1-2 hours, and antibody complexes formed
secondary antibodies were visualized using Enhanced Chemiluminescence reagents
(PicoWest, Pierce, Rockford, IL).
Cell-Cycle Analysis:
Cells were treated with test compounds for the times indicated. Both adherent and nonadherent cells were pooled, washed with cold PBS and fixed in 100% ethanol overnight
at 40C. Ethanol-fixed cells were washed with 1% FBS in PBS, resuspended at 106 cells
per mL, and stained with 10 pg/mL Pl. Stained cells were then incubated with 0.5 pg/mL
RNase A (DNase free; Roche Diagnostics) for 30 minutes at 370C. DNA content was
determined using a Becton-Dickinson flow cytometer (BD FACScan). Cell cycle data
was analyzed using the MODFIT-LT 2.0 program to estimate the percentage of cells in
G1, S and G2/M phases of the cell cycle.
Single-Cell Alkaline Gel Electrophoresis (Comet assay):
Cells were treated with test compounds for indicated times. Alkaline comet assay was
performed as outlined in the Trevigen comet assay protocol using the reagents provided
in the Trevigen kit (Trevigen, Gaithersburg, MD).
Following treatment, cells were
trypsinized, resuspended in PBS at 105 cells/mL and irradiated on ice using a
GammaCell 40 Irradiator with a dose of 10 Gy. Irradiated cells were suspended in liquid
Low Melting Agarose (LMA) at 370C and plated onto a CometSlide (Trevigen). After the
agar solidified, cells were incubated in lysis solution (2.5 M NaCl, 100 mM EDTA, 10mM
Tris base (pH 10), 1%Sodium Lauryl Sarcosine, 0.01% Triton X-100) for an hour at 40C,
followed by incubation with 100 pL of 1 mg/mL Proteinase K (Roche Diagnostics) for an
hour at room temperature. Slides were then treated for 30 minutes with alkaline solution
(0.3 M NaOH, 1 mM EDTA, pH >13), followed by electrophoresis under alkaline
conditions (0.3 M NaOH, 1 mM EDTA). Slides were then dried, stained with SYBR
Green dye, and photographed using a Nikon Eclipse epifluorescence microscope.
Images were analyzed using the Komet 4.2 Single Cell Gel Electrophoresis software
(Kinetic Imaging Limited, U.K.). A total of 100 cells per sample were analyzed, 50 per
duplicate slide. The Olive Tail Moment (OTM), defined as the product of the tail length
and the fraction of total DNA in the tail, was calculated for each cell by the software.
OTM incorporates a measure of both the smallest detectable size of migrating DNA
(reflected in the comet tail length) and the number of relaxed or broken pieces which is
represented by the intensity of DNA in the tail (Olive et al., 1990; Olive and Banath,
2006). Comet data were represented using boxplots plotted using SSC-Stat, a statistical
add-in for Excel.
Boxplot was used to give a graphical representation of the heterogeneity of the comet
data. The box contains the middle 50% of the data; the upper boundary of the box
indicates the
7 5 th
percentile of the data, and the lower boundary the
2 5 th
percentile. The
range of the middle two quartiles is called the interquartile range. The line in the box is
the median. The ends of the "whiskers" on the boxplot extend to a maximum of 1.5 times
the interquartile range. The average OTM values are also indicated on the boxplot.
Percentage decrease in the average OTM was calculated for each treatment relative to
the untreated irradiated control, and significance values were calculated using the MannWhitney statistic (P < 0.001 was considered significant).
Autophagy detection and staining:
Cells were grown in 4-well chamber slides at 105 per well and treated with various drugs.
After treatment, the chambers were washed with PBS.
To detect acidic vesicular
organelles, drug-treated cells were stained with 4 pg/mL acridine orange for 15 min.
Chambers were also co-stained with 4 pg/mL ethidium bromide to detect dying/ dead
cells. Samples were observed under a Nikon Eclipse epifluorescence microscope.
Visualization of autolysosomes: Following treatment with drug, autolysosomes were
labeled with monodansylcadaverine (MDC) by incubating cells grown in chamber slides
with 50pM MDC in PBS at 370C for 15 min. After incubation, cells were washed three
times with PBS and immediately analyzed by fluorescence microscopy for live cell
staining.
Results
E2-7a inhibits the growth of ovarian cancer cells in culture. SKOV-3 and OVCAR-3
cells were treated with a range of doses of E2-7a, cisplatin and chlorambucil for 24
hours and 36 hours. The results are shown in Figures 2.2 and 2.3, and the G150 values
are summarized in Table 2.1.
In SKOV-3 cells, E2-7a was found to be more toxic than cisplatin at both the durations of
treatment. After 24 hours of continuous exposure, E2-7a was over four times as toxic as
cisplatin to SKOV-3 cells, as can be seen from the G150 values. After 36 hours of drug
treatment, the difference in toxicity between the two drugs was less pronounced - the
G150 of cisplatin was 16 pM compared to 6.5 ptM for E2-7ax. In OVCAR-3 cells, a similar
trend was observed - E2-7c was more toxic than cisplatin at both the durations of
treatment. Roughly twice the dose of cisplatin was required to produce a growth
inhibition of 50% compared to E2-7x at both 24 and 36 hours of treatment. The extent of
growth inhibition observed in cells treated with E2-7a was similar in both the cell lines.
These data show that E2-7a is comparable to, if not more effective than, cisplatin in
inducing toxicity in ovarian cancer cells. It is very interesting to note that in both cell lines,
E2-7a was more toxic than chlorambucil, despite the structural similarity of the
molecules. This trend was observed at both durations of treatment.
E2-7a induces S-phase arrest in SKOV-3 and OVCAR-3 cells. SKOV-3 cells were
treated with E2-7c, chlorambucil and cisplatin at 10 pM dose for 24 and 36 hours. Figure
2.4 shows the cell cycle patterns after treatment with the test drugs. In SKOV-3 cells,
E2-7a induced a potent S-phase arrest, which became more pronounced with increased
duration of treatment. Compared to untreated cells which had 21 % cells in S-phase, E27a treated cells had over two thirds of the population arrested in S-phase, which
increased to 76% with 36 hours treatment. Under these conditions, cisplatin treatment at
10 pM did not demonstrate a significant arrest pattern. However, cisplatin at certain
other doses and times of exposure was able to induce S-phase arrest in these cells
(data not shown). Chlorambucil induced a prominent G2/M arrest; over 48% cells
accumulated in G2/M phase after the 24 hour treatment, compared to untreated
population which had 12% cells in G2/M.
In OVCAR-3 cells, as seen in Figure 2.5, a similar trend as in SKOV-3 cells was
observed. E2-7a induced a strong arrest in S-phase, and the treated cells had almost
twice as many cells in S-phase compared to the untreated cells, both at 24 hours and 36
hours after treatment. Cisplatin induced a potent G1 arrest in these cells: -84% and 93%
of the treated cells were arrested in G1 phase after the 24 hour and the 36 hour
treatment respectively, compared to -64% G1 population in the untreated control.
S-phase arrest persists even after drug is removed. We wanted to test if the
differential toxicity between E2-7a and chlorambucil was due to the persistence of the
replication block (S-phase arrest) in E2-7a treated SKOV-3 cells. SKOV-3 cells were
treated with 10piM E2-7a or chlorambucil for 36 hours, and were then allowed to recover
in fresh medium for 28 hours. Treatment with chlorambucil for 36 hours caused a G2/M
arrest, as can be seen in Figure 2.6. On removal of the drug, the G2/M arrest
disappeared over the course of 28 hours, and the cells returned to a distribution similar
to untreated cells after 28 hours of recovery in drug-free medium. Figure 2.7 shows the
cell cycle patterns of cells exposed to E2-7a. E2-7a treatment for 36 hours resulted in an
S-phase arrest as can be seen in the second panel on the top row. Once the drug was
removed and cells grown in fresh medium, the arrest persisted, and after 20 hours of
recovery time, 100% of the cells seemed to be arrested in S-phase, indicating a
persistent replication block. This response was in contrast to that of chlorambucil, where
the G2/M block was effectively removed after drug removal.
E2-7a induces phosphorylation of cdc2 and BRCAI. Various cell cycle checkpoint
proteins were studied to examine whether they were activated to signal cell cycle arrest.
Immunoblotting experiments (Figure 2.8) showed that the phosphorylation of cdc-2 (also
known as CDK1) at Tyrosine 15 (Tyrl5) was seen with chlorambucil, cisplatin and E2-7a
treatment. It must be noted here that the antibody that was used for cdc-2 also
recognizes another cell cycle checkpoint regulator CDK2 that is closely related to cdc-2.
Cdc-2 activation is an essential checkpoint protein that regulates entry into cell cycle.
Phosphorylation of cdc-2 at Tyr1 5 results in inhibiting the progression of cell cycle
predominantly from G2 to M, resulting in cell cycle arrest. The inhibitory phosphorylation
of CDK2 results in G1 arrest and S-phase arrest. The results that are observed are
consistent with the roles of cdc-2 and CDK2 in inducing cell cycle arrest, and all the
three compounds show activation of checkpoint proteins. E2-7a, in particular, induced
S-phase arrest, and this finding is consistent with the phosphorylation of cdc-2 marker.
BRCA1 phosphorylation at Serine 1524 (Ser 524) was also tested with the three drugs.
BRCA1 is a DNA damage recognition protein and is also actuvely involved in cell cycle
regulation. It is phosphorylated at Ser 1524 as a response to DNA damage, and controls
the S-phase checkpoint through CDK2 and G2/M checkpoint via cdc-2. Cisplatin was the
only drug that showed the phosphorylation of BRCA1 at 24 hours, but with 36 hours of
treatment, all the three drugs induced BRCA1 phosphorylation at Ser 524.
E2-7a forms interstrand crosslinks in SKOV-3 cells. SKOV-3 cells treated with
E2-7a were subjected to the modified comet assay (Olive and Banath, 1995; Olive and
Banath, 2006) to determine the presence of interstrand crosslinks (ICLs). Drug treated or
mock treated cells were irradiated with 10 Gy dose to induce random DNA strand
breakage, followed by cell lysis and alkaline unwinding. The presence of ICLs retards the
migration of the irradiated DNA during alkaline electrophoresis, resulting in a reduced
Olive Tail Moment (OTM) compared to the untreated irradiated control. The results are
shown in Figure 2.10 and Table 2.2. The percent decrease in OTM values as seen in
Table 2.2 represents the extent of ICL formation.
We observed that the cells responded to the positive control mechlorethamine (ME) by a
significant decrease in OTM of -45% compared to untreated, indicating that ME was
able to form ICLs in SKOV-3 cells. E2-7a treatment with various doses for six hours also
showed a significant decrease in OTM compared to untreated cells. Doses of 0.5 pM,
1 pM and 2 pM resulted in ~ 20%, 25% and 14% decrease in OTM, respectively. There
was no particular dose response observed in the "extent" of crosslink formation as
quantified by the percent decrease in OTM. Also, E2-7ax did not induce ICL formation as
much as ME did, possibly because of the difference in the kinetics of reaction of the two
compounds.
E2-7a is more toxic to NER deficient XPA cells compared to NER proficient HeLa
cells. In order to examine whether the repair of DNA damage was a player in the
toxicity of E2-7ax, a system of NER deficient and proficient cells (XPA and HeLa,
respectively) were treated with test drugs, and the colony formation ability of the cells
was studied after treatment. The results are shown in Figure 2.10. E2-7a was toxic to
both HeLa and XPA cells, but it was clear that XPA cells were much more sensitive to
the drug than HeLa cells as seen in Figure 2.10. As estimated from the G150 values, XPA
cells were approximately six times more sensitive to E2-7a than HeLa cells. At doses
above 0.5 pM, there were no surviving XPA colonies, whereas ~ 10% of HeLa cells
formed colonies even at 1.25 and 2.5 pM. The two sets of cells were also treated with
chlorambucil, and the results showed a modest differential in toxicity between XPA and
HeLa cells. XPA cells were significantly more sensitive to E2-7aC than to chlorambucil; at
1.25 puM, about 10% of the colonies in chlorambucil treated cells survived, as opposed to
zero survivors in the E2-7a treated cells. The differential in sensitivity between E2-7aX
and chlorambucil in HeLa cells was not as pronounced as in the XPA population. These
results suggest that NER is likely to be involved in the repair of E2-7a lesions that are
produced in these cells.
Cell death caused by E2-7a does not proceed through caspase-3 cleavage
pathway in SKOV-3 cells; E2-7a upregulates level of BcI-2, and does not induce
PARP cleavage. Since E2-7a showed promising cellular toxicity, the next step was to
investigate whether SKOV-3 cells undergo apoptosis, as they do with several DNA
damaging agents such as cisplatin, mechlorethamine and melphalan (Ahn et al., 2004;
Chen et al., 1999; Gibb et al., 1997). SKOV-3 cells were immunoblotted for caspase-3
(the antibody also detects the cleaved form). Results are shown in 2.11. None of the
drug treatments demonstrated the cleavage of caspase-3. With cisplatin, literature
shows apoptosis induction at -80 pM dose in SKOV-3 cells, which probably explains
why we did not see caspase cleavage at 20 pM dose of cisplatin (Gibb et al., 1997). At
20 gM dose of E2-7a, there were no adherent cells remaining on the culture dish after
48 hours of treatment, and it was surprising that the floating dead cells did not display
caspase-3 cleavage. In order to examine whether earlier players in the caspase pathway
were involved, caspase-9 cleavage was also examined - caspase-9 is the earliest
caspase that initiates the process of apoptosis and is upstream of caspase-3. The
results showed the same trend as with caspase-3. No cleavage product was detected
with any of the drug treatments.
Other participants in the classical apoptosis pathway were examined in SKOV-3 cells
(Figure 2.11). Bcl-2 is an anti-apoptotic protein that prevents apoptosis in response to
various stimuli, and downregulation of Bcl-2 levels upon treatment with drug usually
facilitates apoptosis (Newton and Strasser, 1998). Surprisingly, E2-7a at 20 pM dose
upregulated the level of Bcl-2 in these cells. Bcl-2 upregulation has been correlated with
resistance to apoptosis in several systems (Igney and Krammer, 2002; Newton and
Strasser, 1998). The increased level of Bcl-2 with E2-7a. treatment probably explains the
observation that the classical signs of apoptosis were not observed in SKOV-3 cells, as
indicated by the lack of caspase-3 cleavage. In addition, PARP cleavage was not
detected with any of the treatments. PARP is a nuclear enzyme that is involved
predominantly in apoptosis and in DNA repair. It is downstream of caspase-3 in the
apoptosis pathway, and usually gets cleaved by caspase-3 during the process of
apoptosis. We were interested in testing whether PARP cleavage occurred as a
response to drug treatment. From Figure 2.11, it can be seen that PARP cleavage did
not occur, indicating that the classical apoptosis pathway resulting in PARP cleavage
leading to DNA fragmentation was not activated in these cells. Alternative pathways to
cell death seemed to involved, which were investigated further.
E2-7a induces cell death by autophagy. When E2-7a treated SKOV-3 cells were
observed under the microscope, there were no visible signs of the classical apoptosis
pathway, such as nuclear condensation or membrane blebbing, even at doses that
induced over 70% cell death. Instead, several cytoplasmic granular bodies were
observed. This result, in combination with the lack of activation of specific apoptotic
markers, strongly indicated that non-apoptotic death mechanisms might be involved.
Non-apoptotic programmed cell death is principally attributed to autophagy (Reggiori and
Klionsky, 2002). The process of autophagy starts with the autophagosome formation,
leading to the formation of autophagolysosomes (or autolysosomes) by the fusion of
acidic lysosomes with autophagosomes (Hippert et al., 2006). It was hypothesized that
the cytoplasmic granular bodies that were observed were acidic autophagosomes, and
were part of the autophagosome-lysosome degradation pathway. Treated cells were
stained with acridine orange (AO), which fluoresces orange in acidic vesicles, to
visualize acidic autophagolysosomes (AO puncta have been used as a measure of
autophagy (Paglin et al., 2001)). Cells are also co-treated with ethidium bromide (EB) to
detect dead/ dying cells. EB is excluded by viable cells, but can enter cells containing
compromised membranes, and stain the nuclei orange, and hence EB is a marker for
cells that are in the process of dying.
As seen in Figure 2.12 and Figure 2.13, the untreated cells had a background level of
AO fluorescence, indicating the presence of lysosomal compartments. Untreated cells
also excluded EB, and this was confirmed by the absence of orange EB-stained nuclei.
Cisplatin treatments for 12 hours at 5 pM (Figure 2.12) and 24 hours at 2.5 pM (Figure
2.13) looked similar to the untreated cells. E2-7a treatment, on the other hand, markedly
increased the number and size of the acidic compartments in the cell, indicating that
autophagic process may be activated by E2-7ax. The second E2-7a panel in Figure 2.12
also showed several cells with orange nuclei, indicating cell death. It was also seen that
the nuclei were still intact, and did not exhibit signs of fragmentation. Figure 2.13
represents the AO/EB staining at 2.5 pM dose of drug for 24h and this time point showed
a similar trend as the 12h treatment. Very prominent orange punctuate staining in the
cytoplasm was seen with E2-7a treatment. Cisplatin did not induce such staining even at
doses as high as 10 pM (data not shown), but cellular nuclei did start fluorescing orange
with EB staining of cisplatin treated cells, indicating that cell membrane did get
compromised, but even at the 10 ptM dose, cisplatin treated cells did not demonstrate
accumulation of acidic vesicles.
Cells were also stained with MDC, which is a specific marker for late stage
autolysosomes (Biederbick et al., 1995; Klionsky et al., 2008). MDC staining was
performed in addition to AO staining to determine independently whether E2-7a
triggered autophagy. Figure 2.14 shows the results of MDC staining. E2-7C at 2.5 pM
dose for 24 hours induced intense punctuate fluorescent pattern with MDC in the cell,
which is an indicator of autophagic pathway being activated. The untreated cells
displayed an even background green staining with MDC, as did cells treated with 2.5 pM
cisplatin for 24 hours. Taken together, the AO staining and the MDC staining suggest an
autophagic mechanism of cell death with E2-7a treatment.
Discussion
Alkylating agents, in general, have two major impacts on living mammalian cells:
cytotoxicity and delay in cell progression through the cell cycle (Meyn and Murray, 1986).
In the first part of this chapter, the biological outcome of E2-7a action within the
framework of toxicity and cell cycle effects has been studied in ovarian cancer cells. The
remaining chapter deals with the characterization of cell death pathway in SKOV-3 cells
in response to E2-7ax treatment.
The cytotoxicity of E2-7a was tested in cisplatin-resistant ovarian cancer cell lines
OVCAR-3 and SKOV-3 (Hamilton et al., 1983; Morimoto et al., 1993). OVCAR-3 and
SKOV-3 cells were treated with the test compounds cisplatin, chlorambucil, and E2-7aX.
In both the cell lines, E2-7a was found to be more toxic than cisplatin and chlorambucil.
The G150 of cisplatin in SKOV-3 cells treated for 36 hours was 16 pM whereas that of E27a was found to be 6.5 pM. A similar trend was seen with OVCAR-3 cells, with E2-7a
showing promising results in inducing cytotoxicity. E2-7a had almost the same G150 in
OVCAR-3 cells as in SKOV-3 cells, and cisplatin was slightly more toxic to these cells
than to SKOV-3 cells (G150 of 12 pM in OVCAR-3 vs. 16 pM in SKOV-3). The IC50 of
cisplatin from literature studies in SKOV-3 cells is estimated to be ~ 6.7 gM and in
OVCAR-3 cells to be 4.6 pM (Roberts et al., 2005). The difference in the numbers can
be explained by the fact that this study was conducted in a 96-well plate format and the
numbers were estimated using an MTT assay (which is a measure of metabolic activity
of cells), as opposed to growth inhibition by cell counting. However, the trend that
cisplatin is slightly more toxic to OVCAR-3 cells than to SKOV-3 cells as demonstrated
by Roberts et al. (2005) is also observed in the experiments described in this chapter.
When comparing the efficacies of the cisplatin and E2-7c, the fold difference in G150
values, if not the absolute values, might be more useful. At the higher duration of
treatment, E2-7a is twice as effective as cisplatin in inhibiting cell growth by 50% in
SKOV-3 and OVCAR-3 cells under the conditions of our experiments. This study gives
us an initial indication of the therapeutic potential of our compound compared to cisplatin,
currently the most established chemotherapeutic for ovarian cancer.
Compared to chlorambucil, E2-7a is significantly more toxic to both cell lines. As seen
from Table 2.1, the G150 of the control compound chlorambucil is almost four times as
much as E2-7a in SKOV-3 cells. In OVCAR-3 cells, chlorambucil treatment results in a
G150 of 17 piM, over twice as much as that of E2-7ca. Chlorambucil was chosen for
comparison with E2-7a because it contains the same NN-bis-(2-chloroethyl)-aniline
moiety and is expected to form the same types of DNA damage as does E2-7a.
Therefore, the observation that E2-7a is more toxic that chlorambucil implies that the
presence of the estradiol domain tethered to the DNA damaging warhead plays a major
role in the additional toxicity seen with E2-7ax.
E2-7a contains a nitrogen mustard moiety capable of forming monoadducts and
crosslinks (Brookes and Lawley, 1961), and the repair of such kinds of DNA damage can
lead to delay in cell cycle progression. The experiments described in this chapter show
that E2-7a induced a strong S-phase arrest in both SKOV-3 and OVCAR-3 cells,
suggesting that the drug might be creating a replication block in these cells. Such cell
cycle pattern changes were not observed when the compound was used to treat MCF-7
and MBA-MB-231 breast cancer cells (John Marquis, unpublished data). This indicates
that the S-phase arrest pattern is cell line specific, and possibly depends on the extent
and kind of damage occurring in the cell line. Cisplatin induced a strong G1 arrest in
OVCAR-3 cells, and this is in agreement with studies in OVCAR-3 cells treated with
varying doses of cisplatin (Gibb et al., 1997).
Checkpoint protein activation could help explain the observed block in cell cycle in
SKOV-3 cells treated with various test compounds. E2-7a-induced S-phase arrest in
SKOV-3 cells was also accompanied by the phosphorylation of cell cycle checkpoints
proteins cdc-2 and BRCA1. Cell cycle entry into mitotic phase is initiated by the
dephosphorylation of two inhibitory residues Tyr1 5 and Thr1 4 of cdc-2 in G2 phase. The
dephosphorylation is carried out by the cell cycle regulatory protein cdc25c, followed by
activation of cdc-2 - cyclinB1 complex (Zhou and Elledge, 2000). Phosphorylation of
cdc-2 at Tyr15 inhibits the formation of the cdc-2 - cyclinB1 complex, and prevents the
cell cycle from progressing through G2/M phase. Cdc-2 also seems to act as an S-phase
checkpoint protein - elevated levels of phospho-cdc2 (Tyr1 5) are associated with
S-phase arrest after DNA damage in several systems (Barth et al., 1996; Tyagi et al.,
2005). Therefore, the phosphorylation of cdc-2 at Tyr1 5 seems to be a marker for both
S-phase and G2/M phase arrest in cell cycle progression. Another factor to be
considered is that the antibody to phospho-cdc-2 also recognizes endogenous levels of
the phosphorylated checkpoint protein CDK2, which is actively involved in G1 to S
progression and S to G2 progression.
Cdc-2 phosphorylation was seen to occur with treatment with all the three drugs, even
though the treatments arrested cells in different phases of the cell cycle. This can be
explained in one or more ways - (i) the role of cdc-2 in both the G2/M and S-phase
checkpoints can directly explain the results that we observed. If inhibitory
phosphorylation of cdc-2 was detected, it could indicate either S-phase arrest or G2/M
arrest. (ii) The cross-reactivity of the phospho-cdc-2 antibody with phospho-CDK2
implies that the result we obtain could either be cdc-2 or CDK2 activation. Since CDK2
phosphorylation is associated with S-phase arrest, this helps explain why we see the
phosphorylated protein band with E2-7a treatment. These reasons help explain why
chlorambucil and cisplatin are also able to show the presence of phosphorylated protein
in the western blot.
BRCA1 is found to be phosphorylated at Ser1524 in response to several DNA damaging
agents (Deng, 2006), and the phosphorylation of BRCA1 is responsible for controlling
the progression through S-phase. E2-7ca treatment resulted in Ser1524 phosphorylation
of BRCA1, which could explain the S-phase arrest in SKOV-3 cells. BRCA1 is also found
to be involved in controlling the G2/M checkpoint in response to ionizing radiationinduced DNA damage (Narod and Foulkes, 2004; Venkitaraman, 2004) and is found to
be phosphorylated at Ser1524 by the damage sensor protein ATR, as shown by Deng et
al. (2006). BRCA1, similar to cdc-2 is activated in both S phase and G2/M checkpoints,
and this could explain why all the three drugs, which arrested cells in different phases of
the cell cycle, showed phosphorylation of both these proteins. The phospho-proteins
activated by E2-7a are consistent with the pattern of cell cycle arrest observed with the
drug.
Mechanistically, it is very interesting to note that E2-7a and chlorambucil arrest cells in
different phases of the cell cycle. Furthermore, the S-phase arrest by E2-7a persisted
even 28 hours after the drug was removed, whereas following chlorambucil treatment,
cells recovered from the arrest within this time period. This gives rise to several
possibilities, one being that the arrest induced by E2-7a is irreversible, which leads to
cell death and accounts for the differential toxicity between E2-7a and chlorambucil.
Given that the aniline mustard moiety is capable of forming crosslinks, the block to
replication by E2-7a could be attributed to the presence of unrepaired interstrand
crosslinks in the treated cells. This hypothesis was supported by the comet assay, which
was able to detect a modest yet measurable level of cross-linking by E2-7ax. The
nitrogen mustard mechlorethamine (ME) was used as a positive control. There is
evidence from several reports that ME-induced crosslink formation can be easily
detected using the comet assay (Hartley et al., 1991; Sunters et al., 1992). This was the
case in SKOV-3 cells as well, and ME induced a significant decrease in OTM compared
to the control cells, indicative of ICL formation. The three doses of E2-7ax were also able
to show the presence of ICLs, although to a lesser extent than ME. The kinetics of
reaction with DNA and crosslink formation of E2-7a are likely to be different from those
of ME (Essigmann et al., 2001a; Pratt et al., 1994), which probably explains the lower
level of crosslinks observed with E2-7a. Another finding with E2-7a treated cells was
that there was no particular dose response in crosslink formation. Also, several other
doses of E2-7a and longer treatment times were assessed with this technique but
crosslinks could not be detected at these doses and times of treatment (data not shown).
In fact, in many cases, the effect of single strand breaks likely confounded the results of
the assay in such a way that the treated, irradiated cells had a higher tail moment than
the untreated, irradiated cells. Inthis light, it is possible that the tail moments observed in
Table 2.2 and Figure 2.9 represent the net result of crosslinks and single strand breaks,
but the finding that these OTM values are significantly lesser than those of the irradiated
cells provides strong evidence that crosslink formation does occur with E2-7a treatment
(Further direct measurement experiments using Triple Quadrupole Mass Spectrometry
are being conducted to detect and measure directly the levels of monoadducts and
crosslinks in SKOV-3 cells treated with E2-7a).
The next piece of the puzzle in the role of DNA damage in toxicity fell in place through
indirect experimental evidence with HeLa and XPA cells - NER proficient and NER
deficient cell lines, respectively. NER is one of the several repair pathways by means of
which a cell defends itself from exogenous and endogenous DNA damage, and it has
been shown to be the primary repair pathway involved in the removal of cyclobutane
pyrimidine dimers and (6-4) photoproducts (Friedberg et al., 1995). NER has also been
implicated in the repair of bulky adducts formed by alkylating agents such as cisplatin
and melphalan (Furuta et al., 2002; Grant et al., 1998; Reardon et al., 1999; Wu et al.,
2003). XPA protein is the major DNA damage recognition protein in the NER pathway,
and plays a central role in the formation of the excision complex (States et al., 1998).
XPA is required for the initial step of damage recognition and DNA binding (Asahina et
al., 1994; Jones and Wood, 1993). XPA cells are deficient in this key damage
recognition protein, and therefore are an ideal model system of NER deficient cells. XPA
cells are known to be hypersensitive to the DNA damage induced by cisplatin (Stevens
et al., 2008). We were interesting in testing whether DNA damage repair mediated by
NER was involved in the toxicity of E2-7c. Shawn Hiller has performed a similar
experiment in an in vitro system to determine the contribution of NER in repairing E2-7a
- DNA adducts in HeLa whole cell extracts. It was found that the addition of XPA
antibody to a reaction mixture containing Hela cell extracts and either E2-7a-damaged or
UV-damaged pGEM plasmid inhibited the repair of both the plasmids, although by
different extents. The role of XPA in the repair of the damage in the plasmid was
confirmed when the addition of XPA protein to the reaction mix restored the repair of
both E2-7a-damaged and UV-damaged plasmids. This experiment indicates that NER is
one of the key pathways involved in the repair of E2-7a-induced DNA damage.
In my cell culture experimental set-up, I have made a comparison between the
sensitivities of HeLa cells and XPA cells; HeLa cells are NER proficient and contain ~
200,000 XPA protein molecules per cell as compared to zero XPA protein molecules in
the NER deficient cell line (Koberle et al., 2006), and it is, therefore, not surprising that
XPA cells are hypersensitive to the E2-7ca. The clonogenic studies I have conducted do
not rule out the contribution of other repair pathways - in fact, the experiment only
indicates that NER is one of the means by which E2-7ax DNA damage might be repaired.
Several other mechanisms are plausible, such as Base Excision Repair (BER) and
depurination of E2-7a adducts to produce apurinic (or apyrimidinic) (AP) sites which can
be then be processed by AP endonucleases (Friedberg et al., 1981; Lindahl, 1979). My
experiments demonstrate that XPA cells are several folds more sensitive to E2-7a than
HeLa cells, in spite of being only marginally more sensitive than HeLa cells to
chlorambucil, and this result is strongly suggestive of a role for NER in the repair of DNA
lesions associated with E2-7a.
A unique aspect of E2-7a that has not been observed with any of our other rationally
designed drugs was unfolded in the experiments described later in this chapter - E2-7a
induced cell death by regulated cellular self-digestion or autophagy rather than by the
classical apoptotic pathway. Based on morphology, cell death can be classified into
apoptotic, necrotic and autophagic cell death (Zakeri et al., 1995). Apoptotic cell death
is generally characterized by caspase activation, membrane blebbing and DNA
fragmentation (Clarke, 2002). Necrosis is accompanied by cell swelling, plasma
membrane damage, and organelle breakdown (Festjens et al., 2006). The hallmark of
autophagy is the formation of autophagosomes, which eventually fuse with lysosomes to
form autophagolysosomes (also called autolysosomes) (Levine and Klionsky, 2004).
These cell death pathways are not as distinct as they were once though to be and it
appears that they have several common players and closely associated regulatory
pathways (Vandenabeele et al., 2006). In certain cases, mitochondria are also known to
move into autophagic compartments. Thus, mitochondrial dysfunction might be a
common pathway that overlaps between apoptosis and autophagy; in addition, in a
given cell type, different kinds of cell death might co-exist (Zakeri et al., 1995). Many
signals originally studied in the context of apoptosis are now known to induce autophagy,
and the pathways have some common inhibitory signals as well (Botti et al., 2006;
Gozuacik and Kimchi, 2007; Meijer and Codogno, 2006). Anti-apoptotic proteins such as
Bcl-2 family members inhibit the autophagy protein Beclin-1, and consequently inhibit
autophagy (Pattingre et al., 2005).
Although traditionally considered a pro-survival mechanism, autophagy can lead to cell
death in certain settings where it is uncontrollably upregulated (Mizushima et al., 2008).
Also, in situations where tumor cells cannot die by apoptosis upon exposure to metabolic
stress, autophagy might occur to prevent death by necrosis. The main protein involved in
controlling autophagy is the kinase mammalian Target of Rapamycin (mTOR), which is
downstream of the Class Ill phosphotidylinositol-3-kinase (P13K) pathways (Mizushima,
2007). Cytotoxic drugs trigger autophagy, especially in apoptosis-defective or apoptosisresistant cells, and excessive damage and metabolic stress can promote cell death.
Some common autophagy inducers include rapamycin and tamoxifen; 3-methyladenine,
wortmannin and bafilomycin are known to inhibit autophagy (Mizushima, 2007).
E2-7ca induced cell death through autophagy; under the same conditions, cisplatin did
not show any detectable signs of autophagy. The standard markers of apoptosis such as
caspase cleavage, PARP cleavage and DNA fragmentation were not observed with E27ax treatment. Instead, we noticed the appearance of granular bodies in the cytoplasm
after drug treatment, and this prompted us to research further into the characterization of
cell death. Accumulation of acidic vesicles in treated cells suggested that the vesicles
might represent autolysosomes, the lysosomal degradative vesicles characteristic of
autophagy. Punctuate staining observed with autolysosomal marker MDC supports the
theory that the granular bodies observed are autolysosomes.
The finding that E2-7ax induces autophagy gives us a new channel to target apoptosisresistant tumors. The caveat that should be kept in mind, as with any other targeted
therapy, is that there are still many unknowns in the autophagy regulation mechanism
which might determine the ultimate outcome of treatment with an autophagy inducer in a
specific system.
Conclusions
E2-7a has been shown to exhibit properties that make it a favorable drug candidate with
the mechanisms of action that we had intended it to possess (Mitra et al., 2002). In this
chapter, the cytotoxicity and downstream biological response to the compound has been
investigated in ovarian cancer cells. OVCAR-3 and SKOV-3 cells showed similar toxicity
to the compound, and overall, E2-7c was more toxic to the cell lines than the currently
used chemotherapeutic, cisplatin, and was also more toxic to both cell lines than
chlorambucil. E2-7a induced S-phase arrest through the phosphorylation of checkpoint
proteins cdc-2 and BRCA-1, and the cells remained arrested in S-phase several hours
after drug removal. The presence of interstrand crosslinks as detected by comet assay
could explain the persistent S-phase arrest that -was seen with E2-7a. Interstrand
crosslinks can cause a block in the replication, and if left unrepaired, lead to cell death.
The presence of interstrand crosslinks can help understand the basis for increased
cytotoxicity seen with E2-7a compared to the control compound chlorambucil.
In order to address whether DNA damage repair through NER was a factor in E2-7a
toxicity, XPA (NER deficient) and HeLa (NER proficient) cell lines were treated with E27a and the control compound chlorambucil. Although both HeLa and XPA cells were
more sensitive to E2-7a than to chlorambucil, the differential toxicity was much steeper
in the repair deficient XPA cells, indicating that the XPA cell line might not be able to
repair the DNA damage caused by E2-7ca, which in turn results in the increased
sensitivity to the compound. These data indicate that NER might be one of the routes by
which the DNA damage caused by E2-7a is repaired.
Another interesting observation that was made following drug treatment was the
formation of cytoplasmic granular bodies that accumulated as early as eight hours after
treatment. Staining with the dyes AO and MDC revealed that there was a profusion of
lysosomal fusion bodies after drug treatment; the bodies might represent late stage
autophagosomes, and it seemed that cell death proceeded through the autophagic route
of cellular self-digestion. This pathway represents a novel target for E2-7a, and is
especially advantageous in the ovarian cancer setting where several tumors are known
to be apoptosis-resistant.
In conclusion, evidence has been provided in support of the formation of interstrand
crosslinks, which could lead to the replication block which manifests as an S-phase
arrest. Unrepaired DNA damage can result in persistent S-phase arrest, leading to cell
death. E2-7a has been shown to induce cell death through the non-classical
programmed cell death pathway autophagy. We are optimistic about the efficacy of this
compound in a clinical setting.
-Chapter 2-
G150 values of test compounds in SKOV-3 and
OVCAR-3 cells
G150 (in pM)
.
"...
"""...
""...
""..
"""'""......"*
"'"""-"'"-'""""*"""*"
-v
--"--".".""""-"-"""-""""""
- --------------------- - ----------
Treatment
SKOV-3
6h
24h
OVCAR-3
24h
36h
Chlorambucil
22
24
19
17
Cisplatin
19
16
18
12
E2-7a
6.4
9.0
6.5
..............
;.................
........................
......................................
4.0
Table 2.1: G150 values of chlorambucil, cisplatin and E2-7a
in ovarian cancer cells. These values correspond well with
the trends observed in the cytotoxicity curves.
-Chapter 2-
Comet assay detects E2-7a induced crosslinks in
SKOV-3 cells
..............................................................................................................................................................................
Avg
OTM
Percent
Decrease in
OTM
ME 5 pM
6.4
44
E2-7a 0.5 pM
9.2
20
E2-7a 1 pM
8.6
25
E2-7a 2 pM
9.9
14
Treatment
Table 2.2: Comet assay results. OTM values of treated
samples are shown. The percent decrease in OTM
indicates extent of crosslink formation. E2-7a is shown to
induce a modest amount of crosslink formation.
-Chapter 2-
Mechanism of Reaction of E2-7a with DNA
CI
CI
RO
+
N
R
N
CI~
(CI
Aziridinium ion
Bifunctional alkylating agent
CI
NH
_
R
N
Guanine
CI-
Second aziridi nium
ion formation
N
NNH
2
H
N7 guanine adduct
0
0
Guanine
IN>NH
H
2
OH
R= HO
HO
*0Hs
HN
HN
0
N
N NH2
H
G-G crosslink
Figure 2.1: Activation of the aniline mustard of E2-7a
occurs via aziridinium ion formation. Reaction with guanine
results in an N7 guanine monoadduct; formation of a
second aziridinium ion and reaction with guanine results in
a G-G crosslink.
E2-7a treatment is cytotoxic to SKOV-3 cells
following 24 hours and 36 hours of treatment
1001
CHL
-+-CCis
c
80-
-A- E2-7
0
24 hour treatment
60-
D
c40CU
,* 20
0
5
10
15
20
15
20
Dose in uM
100
80
36 hour treatment
60
40
20
0
5
10
Dose in uM
Figure 2.2: Growth inhibition assay in SKOV-3 cells
treated with cisplatin, chlorambucil and E2-7a. Cells were
more sensitive to E2-7a than to chlorambucil or cisplatin.
GI5 values are listed in Table 2.1
E2-7a treatment is cytotoxic to OVCAR-3 cells
following 24 hours and 36 hours of treatment
100
CHL
i
~80
80
+Cis
-A E2-7c
-
60aI)
24 hour treatment
21
24020-
0
5
10
15
20
15
20
Dose in uM
100
80
60
36 hour treatment
40
20
0
5
10
Dose in uM
Figure 2.3: Growth inhibition assay in OVCAR-3 cells
treated with cisplatin, chlorambucil and E2-7ax. Cells were
more sensitive to E2-7ax than to chlorambucil or cisplatin.
G150 values are provided inTable 2.1.
E2-7a arrests SKOV-3 cells in S-phase
UNTREATED
13
Cisplatin 24h
6
Cisplatin 36h
0
-
25
21
73
75
CHL36h
CHL24h
23
G1
42
48
42
G2/M
E2-7a 24h
E2-7a 36h
0
0
24
33
66
76
Figure 2.4: Cell cycle patterns in SKOV-3 cells treated
with 10 pM dose of cisplatin, chlorambucil and E2-7ax.
E2-7ax induces a potent S-phase arrest, whereas
chlorambucil induces a G2/M arrest. There was no striking
arrest pattern observed with cisplatin under these
conditions
E2-7a arrests cells in S-phase in OVCAR-3 cells
UNT 24h
14
65
Cisplatin 48h
Cisplatin 24h
7
16
31
93
84
G2/M
E2-7a 24h
8
40
E2-7a 48h
8
52
48
Figure 2.5: Cell cycle patterns in OVCAR-3 cells treated
with 10 pM dose of cisplatin and E2-7ax. E2-7a induces a
potent S-phase arrest, whereas cisplatin is shown to halt
cells in G1.
Chlorambucil treated cells recover from G2/M
arrest once drug is removed
UNTREATED
Oh post treatment
15
43
21
45
12
8h post treatment
12h post treatment
32
G1
20
59
59
G2/M
20h post treatment
28h post treatment
17
17
64
Figure 2.6: Cells treated for 36 hours with 10 pM
chlorambucil and allowed to recover for 28 hours in fresh
medium. After 28h, cell cycle patterns are similar to those
of untreated cells.
C atr2-
E2-7a treated cells do not recover from S-phase
arrest after drug is removed
UNTREATED
Oh post treatment
2
15
21
57
8h post treatment
12h post treatment
15
35
41
45
G2/M
58
20h post treatment
28h post treatment
100
100
Figure 2.7: Cells treated for 36 hours with 10 ptM E2-7ca
and allowed to recover for 28 hours in fresh medium. After
28 hours, treated cells are still accumulated in S-phase.
-Chapter 2-
E2-7a induces activation of checkpoint proteins
in SKOV-3 cells
24 hour treatment
1
2
3
4
6
5
p-cdc2
Actin
24 hour treatment
1
2
3
Lane
4
5
6
Treatment
Untreated
2
Cis 20M
p-BRCA1
3
CHL 10M
Actin
4
CHL 20pM
5
E2-7ax 1OpM
6
E2-7ca 20M
36 hour treatment
1
2
3
4
5
6
p-BRCA1
Actin
Figure 2.8: Checkpoint proteins activated upon treatment
with test drugs. Cdc-2 and BRCA1 are activated by E2-7a.
100
-Chapter 2-
Comet assay indicates presence of interstrand
crosslinks after E2-7a treatment
OTM vs. Treatment (6h)
20
16
# Treatment
E12
Untreated
Irradiated 10 Gy
ME 5 gM+Irr
E2-7a 0.5 pM+Irr
E2-7cc 1.0 pM+Irr
E2-7o 2.0 pM+Irr
08
0
1
2
3
4
5
6
Figure 2.9: Boxplot showing distribution of tail moment in
SKOV-3 cells. Numbers above the boxes represent
average OTM value. Both ME and E2-7a treatments
(starred) result ina significant decrease in OTM compared
to the irradiated control sample (P<0.001) . The OTM
values are tabulated inTable 2.2.
101
XPA cells are more sensitive to E2-7a than HeLa
cells
Clonogenic survival: XPA vs. HeLa
100
----- ------------
'A
10
Cl)
U)
Cu
4-'
c
U)
0~
HeLa -E2-7ca
-0-U-
1
0.1
XPA -E2-7a
-
-A
-
HeLa-CHL
-
-+
-
XPA-CHL
0.01
0
1.5
1
0.5
2
2.5
Dose in uM
Treatment
G1
50 in_M
HeLa-E2-7a
0.55
XPA-E2-7ca
0.08
HeLa-CHL
1.32
XPA-CHL
0.44
Figure 2.10: Clonogenic survival response of XPA and
HeLa cells to drugs after 24 hour treatment, followed by
recovery and colony formation. XPA cells are much more
sensitive to E2-7a than HeLa cells. Chlorambucil induces
a marginal increased sensitivity in XPA compared to HeLa
cells.
102
Apoptotic markers are not activated with E2-7a
treatment
48 hour treatment
1
2
3
4
5
6
Caspase-3
Caspase-9
Actin
36 hour treatment
1
2
3
4
5
BcI-2
6
Lane
Treatment
1
2
3
Untreated
Cis 20pM
CHL 10pM
4
CHL20pM
5
6
E2-7o 1OpM
E2-7a 20pM
PARP
Actin
Figure 2.11: Western blot showing apoptotic markers.
Caspase-3, caspase-9 or PARP cleavage are not
observed with E2-7ax treatment. Bcl-2 levels seem to be
upregulated in response to E2-7a treatment.
103
-Chapter 2-
AO/ EB staining of live SKOV-3 cells (treatment
for 12 hours a t 5M dos
Figure 2.12: Treatments as indicated. AO can be seen as
punctuate orange staining inacidic compartments and
green stain inthe nuclei of live cells. Orange nuclei
represent staining by EB inmembrane compromised cells.
104
11 _ _ , __
__ - -1- --
..
....
..
......
..
.......................
. ......
1. _. ,,,......
....
-Chapter 2-
AO/ EB staining of live SKOV-3 cells (treatment
for 24 hours at 2.5 M dos
TR
I"I I",\TIIo
Figure 2.13: Treatments as indicated. AO can be seen as
punctuate orange staining inacidic compartments and
green stain inthe nuclei of live cells. Orange nuclei
represent staining by EB inmembrane compromised cells.
105
................
.....
........
...
...
......
..
-Chapter 2-
MDC staining of live SKOV-3 cells (treatment
for 24 hours at 2.5 pM dose)
GCI IA TVIN
Figure 2.14: Treatments as indicated. MDC can be seen
as punctuate green staining in E2-7ca treated cells and as
a diffuse green stain inuntreated and cisplatin treated cells.
106
Reference List
1.
Abend M (2003) Reasons to reconsider the significance of apoptosis for cancer
therapy. Int J Radiat Biol 79:927-941
2.
Agarwal R, Kaye SB (2003) Ovarian cancer: strategies for overcoming resistance
to chemotherapy. Nat Rev Cancer 3:502-516
3.
Ahn HJ, Kim YS, Kim JU, Han SM, Shin JW, Yang HO (2004) Mechanism of taxolinduced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem
91:1043-1052
4.
Ali S, Metzger D, Bornert JM, Chambon P (1993) Modulation of transcriptional
activation by ligand-dependent phosphorylation of the human oestrogen receptor
A/B region. EMBO J 12:1153-1160
5.
American Cancer Society (2008) Cancer Facts and Figures.
6.
Arai M, Utsunomiya J, Miki Y (2004) Familial breast and ovarian cancers. Int J Clin
Oncol 9:270-282
7.
Araujo SJ, Nigg EA, Wood RD (2001) Strong functional interactions of TFIIH with
XPC and XPG in human DNA nucleotide excision repair, without a preassembled
repairosome. Mol Cell Biol 21:2281-2291
8.
Argento M, Hoffman P, Gauchez AS (2008) Ovarian cancer detection and
treatment: current situation and future prospects. Anticancer Res 28:3135-3138
107
9.
Armitage P, Doll (1954) The age distribution of cancer and a multi-stage theory of
carcinogenesis. Br J Cancer 8:1-12
10.
Asahina H, Kuraoka I, Shirakawa M, Morita EH, Miura N, Miyamoto I, Ohtsuka E,
Okada Y, Tanaka K (1994) The XPA protein is a zinc metalloprotein with an ability
to recognize various kinds of DNA damage. Mutat Res 315:229-237
11.
Balcome S, Park S, Quirk Dorr DR, Hafner L, Phillips L, Tretyakova N (2004)
Adenine-containing DNA-DNA cross-links of antitumor nitrogen mustards. Chem
Res Toxicol 17:950-962
12.
Bandera CA, Ye B, Mok SC (2003) New technologies for the identification of
markers for early detection of ovarian cancer. Curr Opin Obstet Gynecol 15:51-55
13. Bardin A, Hoffmann P, Boulle N, Katsaros D, Vignon F, Pujol P, Lazennec G (2004)
Involvement of estrogen receptor beta in ovarian carcinogenesis. Cancer Res
64:5861-5869
14.
Barth H, Hoffmann 1,Klein S, Kaszkin M, Richards J, Kinzel V (1996) Role of
cdc25-C phosphatase in the immediate G2 delay induced by the exogenous
factors epidermal growth factor and phorbolester. J Cell Physiol 168:589-599
15.
Bast RC, Jr., Klug TL, St JE, Jenison E, Niloff JM, Lazarus H, Berkowitz RS,
Leavitt T, Griffiths CT, Parker L, Zurawski VR, Jr., Knapp RC (1983) A
radioimmunoassay using a monoclonal antibody to monitor the course of epithelial
ovarian cancer. N Engl J Med 309:883-887
16.
Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate
ribonuclease in the initiation step of RNA interference. Nature 409:363-366
108
17.
Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a
specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3-14
18.
Bishop JM (1991) Molecular themes in oncogenesis. Cell 64:235-248
19.
Biswas DK, Singh S, Shi Q, Pardee AB, Iglehart JD (2005) Crossroads of estrogen
receptor and NF-kappaB signaling. Sci STKE 2005:e27
20.
Bonadonna G, Brusamolino E, Valagussa P, Rossi A, Brugnatelli L, Brambilla C,
De LM, Tancini G, Bajetta E, Musumeci R,Veronesi U (1976) Combination
chemotherapy as an adjuvant treatment in operable breast cancer. N Engl J Med
294:405-410
21.
Borini A, Rebellato E (2008) Focus on breast and ovarian cancer. Placenta 29
Suppl B:184-190
22. Botti J, Djavaheri-Mergny M, Pilatte Y, Codogno P (2006) Autophagy signaling and
the cogwheels of cancer. Autophagy 2:67-73
23. Boulikas T, Vougiouka M (2004) Recent clinical trials using cisplatin, carboplatin
and their combination chemotherapy drugs (review). Oncol Rep 11:559-595
24.
Bowler J, Lilley TJ, Pittam JD, Wakeling AE (1989) Novel steroidal pure
antiestrogens. Steroids 54:71-99
25. Brandenberger AW, Lebovic DI, Tee MK, Ryan IP,Tseng JF, Jaffe RB, Taylor RN
(1999) Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial
and endometriosis-derived stromal cells. Mol Hum Reprod 5:651-655
109
26.
Brandenberger AW, Tee MK, Jaffe RB (1998) Estrogen receptor alpha (ER-alpha)
and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma
and ovarian cancer cell lines: down-regulation of ER-beta in neoplastic tissues. J
Clin Endocrinol Metab 83:1025-1028
27.
Brookes, Lawley PD (1961) The reaction of mono- and di-functional alkylating
agents with nucleic acids. Biochem J 80:496-503
28.
Brown JM, Attardi LD (2005) The role of apoptosis in cancer development and
treatment response. Nat Rev Cancer 5:231-237
29.
Brown SJ, Kellett PJ, Lippard SJ (1993) lxrl, a yeast protein that binds to
platinated DNA and confers sensitivity to cisplatin. Science 261:603-605
30.
Bruhn, Toney JH, Lippard SJ (1990) Biological processing of DNA modified by
platinum compounds., 38 edn
31.
Bucourt R, Vignau M, Torelli V (1978) New biospecific adsorbents for the
purification of estradiol receptor. J Biol Chem 253:8221-8228
32.
Burger RA (2007) Experience with bevacizumab in the management of epithelial
ovarian cancer. J Clin Oncol 25:2902-2908
33.
Chabner BA, Longo DL (2009) Cancer Chemotherapy and Biotherapy
34.
Chabner BA, Roberts TG, Jr. (2005) Timeline: Chemotherapy and the war on
cancer. Nat Rev Cancer 5:65-72
35.
Chatterjee M, Mohapatra S, lonan A, Bawa G, li-Fehmi R, Wang X, Nowak J, Ye B,
Nahhas FA, Lu K, Witkin SS, Fishman D, Munkarah A, Morris R, Levin NK, Shirley
110
112
NN, Tromp G,Abrams J, Draghici S, Tainsky MA (2006) Diagnostic markers of
ovarian cancer by high-throughput antigen cloning and detection on arrays. Cancer
Res 66:1181-1190
36. Chen Z, Fadiel A, Jia JF, Sakamoto H, Carbone R, Naftolin F (1999) Induction of
apoptosis and suppression of clonogenicity of ovarian carcinoma cells with
estrogen mustard. Cancer 85:2616-2622
37. Clark (2006) Molecular targeted drugs and growth factor receptor inhibitors. In:
Chabner BA, Longo DL (eds)
38. Clarke PG (2002) Apoptosis: from morphological types of cell death to interacting
pathways. Trends Pharmacol Sci 23:308-309
39. Clinton GM, Hua W (1997) Estrogen action in human ovarian cancer. Crit Rev
Oncol Hematol 25:1-9
40. Coezy E, Borgna JL, Rochefort H (1982) Tamoxifen and metabolites in MCF7 cells:
correlation between binding to estrogen receptor and inhibition of cell growth.
Cancer Res 42:317-323
41.
Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS (1997) Tissue
distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and
estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and
ERalpha-knockout mouse. Endocrinology 138:4613-4621
42. Cowley SM, Hoare S, Mosselman S, Parker MG (1997) Estrogen receptors alpha
and beta form heterodimers on DNA. J Biol Chem 272:19858-19862
111
43.
Cunat S, Hoffmann P, Pujol P (2004) Estrogens and epithelial ovarian cancer.
Gynecol Oncol 94:25-32
44.
DaSilva JN, van Lier JE (1990) Synthesis and structure-affinity of a series of 7
alpha-undecylestradiol derivatives: a potential vector for therapy and imaging of
estrogen-receptor-positive cancers. J Med Chem 33:430-434
45.
De BK, Vanden BW, Haegeman G (2006) Cross-talk between nuclear receptors
and nuclear factor kappaB. Oncogene 25:6868-6886
46.
Deng CX (2006) BRCA1: cell cycle checkpoint, genetic instability, DNA damage
response and cancer evolution. Nucleic Acids Res 34:1416-1426
47.
DeVita VT, Hellman S, Rosenberg SA (1997) Princples and Practice of Oncology,
5 edn
48.
Donahue BA, Augot M, Bellon SF, Treiber DK, Toney JH, Lippard SJ, Essigmann
JM (1990) Characterization of a DNA damage-recognition protein from mammalian
cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the
anticancer drug cisplatin. Biochemistry 29:5872-5880
49.
Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M, Samudio I, Kladde MP,
Vyhlidal C, Safe S (1999) Mechanisms of transcriptional activation of bcl-2 gene
expression by 17beta-estradiol in breast cancer cells. J Biol Chem 274:3209932107
50.
Driscoll MD, Sathya G, Muyan M, Klinge CM, Hilf R, Bambara RA (1998)
Sequence requirements for estrogen receptor binding to estrogen response
elements. J Biol Chem 273:29321-29330
112
'1K) K)
51.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A,Weber K, Tuschl T (2001)
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian
cells. Nature 411:494-498
52.
Essigmann JM, Rink SM, Park HJ, Croy RG (2001b) Design of DNA damaging
agents that hijack transcription factors and block DNA repair. Adv Exp Med Biol
500:301-313
53. Essigmann, Rink SM, Park HJ, Croy RG (2001a) Design of DNA damaging agents
that hijack transcription factors and block DNA repair. In: Dansette (ed) Biological
Reactive Intermediates VI, 6 edn
54. Festjens N, Vanden BT, Vandenabeele P (2006) Necrosis, a well-orchestrated
form of cell demise: signalling cascades, important mediators and concomitant
immune response. Biochim Biophys Acta 1757:1371-1387
55.
Fliss AE, Benzeno S, Rao J, Caplan AJ (2000) Control of estrogen receptor ligand
binding by Hsp90. J Steroid Biochem Mol Biol 72:223-230
56. Foulds (1954) The experimental study of tumor progression: a review. Cancer Res
14:327-339
57. Friedberg EC, Bonura T, Love JD, McMillan S, Radany EH, Schultz RA (1981) The
repair of DNA damage: recent developments and new insights. J Supramol Struct
Cell Biochem 16:91-103
58. Friedberg EC, Walker G, Siede W (1995) DNA repair and mutagenesis
113
59.
Fu XD, Simoncini T (2008) Extra-nuclear signaling of estrogen receptors. IUBMB
Life 60:502-510
60.
Furuta T, Ueda T, Aune G, Sarasin A, Kraemer KH, Pommier Y (2002)
Transcription-coupled nucleotide excision repair as a determinant of cisplatin
sensitivity of human cells. Cancer Res 62:4899-4902
61.
Gibb RK, Taylor DD, Wan T, O'Connor DM, Doering DL, Gercel-Taylor C (1997)
Apoptosis as a measure of chemosensitivity to cisplatin and taxol therapy in
ovarian cancer cell lines. Gynecol Oncol 65:13-22
62.
Gozuacik D, Kimchi A (2007) Autophagy and cell death. Curr Top Dev Biol 78:217245
63.
Grant DF, Bessho T, Reardon JT (1998) Nucleotide excision repair of melphalan
monoadducts. Cancer Res 58:5196-5200
64. Green S, Walter P, Greene G, Krust A, Goffin C, Jensen E, Scrace G, Waterfield M,
Chambon P (1986) Cloning of the human oestrogen receptor cDNA. J Steroid
Biochem 24:77-83
65.
Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J (1986) Sequence and
expression of human estrogen receptor complementary DNA. Science 231:11501154
66.
Greenlee RT, Murray T, Bolden S, Wingo PA (2000) Cancer statistics, 2000. CA
Cancer J Clin 50:7-33
114
67. Haapala E, Hakala K, Jokipelto E, Vilpo J, Hovinen J (2001) Reactions of N,Nbis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'deoxyguanosine. Chem Res Toxicol 14:988-995
68. Hall JM, McDonnell DP (1999) The estrogen receptor beta-isoform (ERbeta) of the
human estrogen receptor modulates ERalpha transcriptional activity and is a key
regulator of the cellular response to estrogens and antiestrogens. Endocrinology
140:5566-5578
69.
Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, WhangPeng J, Rogan AM, Green WR, Ozols RF (1983) Characterization of a human
ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors.
Cancer Res 43:5379-5389
70. Hamilton TC, Young RC, Ozols RF (1984) Experimental model systems of ovarian
cancer: applications to the design and evaluation of new treatment approaches.
Semin Oncol 11:285-298
71.
Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70
72.
Hartley JA, Berardini MD, Souhami RL (1991) An agarose gel method for the
determination of DNA interstrand crosslinking applicable to the measurement of the
rate of total and "second-arm" crosslink reactions. Anal Biochem 193:131-134
73. He Z, Ingles CJ (1997) Isolation of human complexes proficient in nucleotide
excision repair. Nucleic Acids Res 25:1136-1141
74. Helleday T, Petermann E, Lundin C, Hodgson B, Sharma RA (2008) DNA repair
pathways as targets for cancer therapy. Nat Rev Cancer 8:193-204
115
75.
Hippert MM, O'Toole PS, Thorburn A (2006) Autophagy in cancer: good, bad, or
both? Cancer Res 66:9349-9351
76. Ho SM (2003) Estrogen, progesterone and epithelial ovarian cancer. Reprod Biol
Endocrinol 1:73
77. Htun H, Holth LT, Walker D, Davie JR, Hager GL (1999) Direct visualization of the
human estrogen receptor alpha reveals a role for ligand in the nuclear distribution
of the receptor. Mol Biol Cell 10:471-486
78. Hua W, Christianson T, Rougeot C, Rochefort H,Clinton GM (1995a) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
79.
Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995b) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
80. Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995c) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
81.
Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995d) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
82. Huang A, Kaley G (2004) Gender-specific regulation of cardiovascular function:
estrogen as key player. Microcirculation 11:9-38
116
83. Huang JC, Zamble DB, Reardon JT, Lippard SJ, SancarA (1994) HMG-domain
proteins specifically inhibit the repair of the major DNA adduct of the anticancer
drug cisplatin by human excision nuclease. Proc Natl Acad Sci U S A 91:1039410398
84. Hurley LH (2002) DNA and its associated processes as targets for cancer therapy.
Nat Rev Cancer 2:188-200
85.
Igney FH, Krammer PH (2002) Death and anti-death: tumour resistance to
apoptosis. Nat Rev Cancer 2:277-288
86. Jacobs I, Bast RC, Jr. (1989) The CA 125 tumour-associated antigen: a review of
the literature. Hum Reprod 4:1-12
87. Jaffe N, Frei E, Ill, Traggis D, Bishop Y (1974) Adjuvant methotrexate and
citrovorum-factor treatment of osteogenic sarcoma. N Engl J Med 291:994-997
88. Jakowlew SB, Breathnach R,Jeltsch JM, Masiakowski P, Chambon P (1984)
Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell
line MCF-7. Nucleic Acids Res 12:2861-2878
89. Jemal A, Siegel R,Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer
statistics, 2008. CA Cancer J Clin 58:71-96
90. Jones CJ, Wood RD (1993) Preferential binding of the xeroderma pigmentosum
group A complementing protein to damaged DNA. Biochemistry 32:12096-12104
91.
Kim DH, Rossi JJ (2007) Strategies for silencing human disease using RNA
interference. Nat Rev Genet 8:173-184
117
92.
King WJ, Greene GL (1984) Monoclonal antibodies localize oestrogen receptor in
the nuclei of target cells. Nature 307:745-747
93.
Klein-Hitpass L, Ryffel GU, Heitlinger E, Cato AC (1988) A 13 bp palindrome is a
functional estrogen responsive element and interacts specifically with estrogen
receptor. Nucleic Acids Res 16:647-663
94.
Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M,
Baehrecke EH, Bahr BA, Ballabio A, Bamber BA, Bassham DC, Bergamini E, Bi X,
Biard-Piechaczyk M, Blum JS, Bredesen DE, Brodsky JL, Brumell JH, Brunk UT,
Bursch W, Camougrand N, Cebollero E, Cecconi F, Chen Y, Chin LS, Choi A, Chu
CT, Chung J, Clarke PG, Clark RS, Clarke SG, Clave C, Cleveland JL, Codogno P,
Colombo MI, Coto-Montes A, Cregg JM, Cuervo AM, Debnath J, Demarchi F,
Dennis PB, Dennis PA, Deretic V, Devenish RJ, Di SF, Dice JF, Difiglia M, neshKumar S, Distelhorst CW, Djavaheri-Mergny M, Dorsey FC, Droge W, Dron M,
Dunn WA, Jr., Duszenko M, Eissa NT, Elazar Z, Esclatine A, Eskelinen EL, Fesus
L, Finley KD, Fuentes JM, Fueyo J, Fujisaki K, Galliot B, Gao FB, Gewirtz DA,
Gibson SB, Gohla A, Goldberg AL, Gonzalez R, Gonzalez-Estevez C, Gorski S,
Gottlieb RA, Haussinger D, He YW, Heidenreich K, Hill JA, Hoyer-Hansen M, Hu X,
Huang WP, Iwasaki A, Jaattela M, Jackson WT, Jiang X, Jin S, Johansen T, Jung
JU, Kadowaki M, Kang C, Kelekar A, Kessel DH, Kiel JA, Kim HP, Kimchi A,
Kinsella TJ, Kiselyov K, Kitamoto K, Knecht E, Komatsu M, Kominami E, Kondo S,
Kovacs AL, Kroemer G, Kuan CY, Kumar R, Kundu M, Landry J, Laporte M, Le W,
Lei HY, Lenardo MJ, Levine B, Lieberman A, Lim KL, Lin FC, Liou W, Liu LF,
Lopez-Berestein G, Lopez-Otin C, Lu B, Macleod KF, Malorni W, Martinet W,
Matsuoka K, Mautner J, Meijer AJ, Melendez A, Michels P, Miotto G, Mistiaen WP,
Mizushima N, Mograbi B, Monastyrska I, Moore MN, Moreira PI, Moriyasu Y, Motyl
118
T, Munz C, Murphy LO, Naqvi NI, Neufeld TP, Nishino I, Nixon RA, Noda T,
Nurnberg B, Ogawa M, Oleinick NL, Olsen LJ, Ozpolat B, Paglin S, Palmer GE,
Papassideri 1,Parkes M, Perlmutter DH, Perry G, Piacentini M, Pinkas-Kramarski
R, Prescott M, Proikas-Cezanne T, Raben N, Rami A, Reggiori F, Rohrer B,
Rubinsztein DC, Ryan KM, Sadoshima J, Sakagami H, Sakai Y, Sandri M,
Sasakawa C, Sass M, Schneider C, Seglen PO, Seleverstov 0, Settleman J,
Shacka JJ, Shapiro IM, Sibirny A, Silva-Zacarin EC, Simon HU, Simone C,
Simonsen A, Smith MA, Spanel-Borowski K, Srinivas V, Steeves M, Stenmark H,
Stromhaug PE, Subauste CS, Sugimoto S, Sulzer D, Suzuki T, Swanson MS,
Tabas I, Takeshita F, Talbot NJ, Talloczy Z, Tanaka K, Tanaka K, Tanida 1,Taylor
GS, Taylor JP, Terman A, Tettamanti G, Thompson CB, Thumm M, Tolkovsky AM,
Tooze SA, Truant R, Tumanovska LV, Uchiyama Y, Ueno T, Uzcategui NL, van dK,
I, Vaquero EC, Vellai T, Vogel MW, Wang HG, Webster P, Wiley JW, Xi Z, Xiao G,
Yahalom J, Yang JM, Yap G, Yin XM, Yoshimori T, Yu L, Yue Z, Yuzaki M,
Zabirnyk 0, Zheng X, Zhu X, Deter RL (2008) Guidelines for the use and
interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy
4:151-175
95.
Koberle B, Roginskaya V, Wood RD (2006) XPA protein as a limiting factor for
nucleotide excision repair and UV sensitivity in human cells. DNA Repair (Amst)
5:641-648
96.
Kohn KW (1996) Beyond DNA cross-linking: history and prospects of DNAtargeted cancer treatment--fifteenth Bruce F. Cain Memorial Award Lecture.
Cancer Res 56:5533-5546
119
97.
Kohn KW, Hartley JA, Mattes WB (1987) Mechanisms of DNA sequence selective
alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res
15:10531-10549
98.
Krasner C (2007) Aromatase inhibitors in gynecologic cancers. J Steroid Biochem
Mol Biol 106:76-80
99.
Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson
JA (1997) Comparison of the ligand binding specificity and transcript tissue
distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870
100. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996) Cloning of
a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A
93:5925-5930
101. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van
der BB, Gustafsson JA (1998a) Interaction of estrogenic chemicals and
phytoestrogens with estrogen receptor beta. Endocrinology 139:4252-4263
102. Kuiper GG, Shughrue PJ, Merchenthaler I, Gustafsson JA (1998b) The estrogen
receptor beta subtype: a novel mediator of estrogen action in neuroendocrine
systems. Front Neuroendocrinol 19:253-286
103. Kundu GC, Schullek JR, Wilson IB(1994) The alkylating properties of chlorambucil.
Pharmacol Biochem Behav 49:621-624
104. Kurreck J (2009) RNA interference: from basic research to therapeutic applications.
Angew Chem Int Ed EngI 48:1378-1398
120
105. Kushner PJ, Agard D, Feng WJ, Lopez G,Schiau A, Uht R, Webb P, Greene G
(2000a) Oestrogen receptor function at classical and alternative response
elements. Novartis Found Symp 230:20-26
106. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P
(2000b) Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311317
107. Lahooti H,White R, Danielian PS, Parker MG (1994) Characterization of liganddependent phosphorylation of the estrogen receptor. Mol Endocrinol 8:182-188
108. Langdon SP, Hirst GL, Miller EP, Hawkins RA, Tesdale AL, Smyth JF, Miller WR
(1994) The regulation of growth and protein expression by estrogen in vitro: a
study of 8 human ovarian carcinoma cell lines. J Steroid Biochem Mol Biol 50:131135
109. Langdon SP, Ritchie A, Young K, Crew AJ, Sweeting V, Bramley T, Hillier S,
Hawkins RA, Tesdale AL, Smyth JF, . (1993) Contrasting effects of 17 betaestradiol on the growth of human ovarian carcinoma cells in vitro and in vivo. Int J
Cancer 55:459-464
110. Lau KM, Mok SC, Ho SM (1999) Expression of human estrogen receptor-alpha
and -beta, progesterone receptor, and androgen receptor mRNA in normal and
malignant ovarian epithelial cells. Proc Natl Acad Sci U S A 96:5722-5727
111. Lawley PD (1966) Effects of some chemical mutagens and carcinogens on nucleic
acids. Prog Nucleic Acid Res Mol Biol 5:89-131
121
112. Lazennec G (2006) Estrogen receptor beta, a possible tumor suppressor involved
in ovarian carcinogenesis. Cancer Lett 231:151-157
113. Le GP, Montano MM, Schodin DJ, Katzenellenbogen BS (1994) Phosphorylation of
the human estrogen receptor. Identification of hormone-regulated sites and
examination of their influence on transcriptional activity. J Biol Chem 269:44584466
114. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular
mechanisms and biological functions of autophagy. Dev Cell 6:463-477
115. Li M, HERTZ R, BERGENSTAL DM (1958) Therapy of choriocarcinoma and
related trophoblastic tumors with folic acid and purine antagonists. N Engl J Med
259:66-74
116. Liberman RG, Tannenbaum SR, Hughey BJ, Shefer RE, Klinkowstein RE, Prakash
C, Harriman SP, Skipper PL (2004) An interface for direct analysis of (14)c in
nonvolatile samples by accelerator mass spectrometry. Anal Chem 76:328-334
117. Lindahl T (1979) DNA glycosylases, endonucleases for apurinic/apyrimidinic sites,
and base excision-repair. Prog Nucleic Acid Res Mol Biol 22:135-192
118. Lindgren PR, Cajander S, Backstrom T, Gustafsson JA, Makela S, Olofsson JI
(2004) Estrogen and progesterone receptors in ovarian epithelial tumors. Mol Cell
Endocrinol 221:97-104
119. Lindor NM, Petersen GM, Hadley DW, Kinney AY, Miesfeldt S, Lu KH, Lynch P,
Burke W, Press N (2006) Recommendations for the care of individuals with an
122
inherited predisposition to Lynch syndrome: a systematic review. JAMA 296:15071517
120. Lukanova A, Kaaks R (2005) Endogenous hormones and ovarian cancer:
epidemiology and current hypotheses. Cancer Epidemiol Biomarkers Prev 14:98107
121. MacLeod MC, Powell KL, Tran N (1995) Binding of the transcription factor, Sp1, to
non-target sites in DNA modified by benzo[a]pyrene diol epoxide. Carcinogenesis
16:975-983
122. Maggi A, Ciana P, Belcredito S, Vegeto E (2004) Estrogens in the nervous system:
mechanisms and nonreproductive functions. Annu Rev Physiol 66:291-313
123. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K,
Blumberg B, Kastner P, Mark M, Chambon P, Evans RM (1995) The nuclear
receptor superfamily: the second decade. Cell 83:835-839
124. Masiakowski P, Breathnach R, Bloch J, Gannon F, Krust A, Chambon P (1982)
Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human
breast cancer cell line. Nucleic Acids Res 10:7895-7903
125. Masood S, Heitmann J, Nuss RC, Benrubi GI (1989) Clinical correlation of
hormone receptor status in epithelial ovarian cancer. Gynecol Oncol 34:57-60
126. May FE, Westley BR (1988) Identification and characterization of estrogenregulated RNAs in human breast cancer cells. J Biol Chem 263:12901-12908
123
127. May FE, Westley BR (1995) Estrogen regulated messenger RNAs in human breast
cancer cells. Biomed Pharmacother 49:400-414
128. McA'Nulty MM, Lippard SJ (1996) The HMG-domain protein lxrl blocks excision
repair of cisplatin-DNA adducts in yeast. Mutat Res 362:75-86
129. McA'Nulty MM, Whitehead JP, Lippard SJ (1996) Binding of lxrl, a yeast HMGdomain protein, to cisplatin-DNA adducts in vitro and in vivo. Biochemistry
35:6089-6099
130. Meijer AJ, Codogno P (2006) Signalling and autophagy regulation in health, aging
and disease. Mol Aspects Med 27:411-425
131. Merajver SD, Pham TM, Caduff RF, Chen M, Poy EL, Cooney KA, Weber BL,
Collins FS, Johnston C, Frank TS (1995) Somatic mutations in the BRCA1 gene in
sporadic ovarian tumours. Nat Genet 9:439-443
132. Meyn, Murray D (1986) Cell cycle Effects of alkylating agents.
133. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu
Q, Cochran C, Bennett LM, Ding W, . (1994) A strong candidate for the breast and
ovarian cancer susceptibility gene BRCA1. Science 266:66-71
134. Mitra K, Marquis JC, Hillier SM, Rye PT, Zayas B, Lee AS, Essigmann JM, Croy
RG (2002) A rationally designed genotoxin that selectively destroys estrogen
receptor-positive breast cancer cells. J Am Chem Soc 124:1862-1863
135. Mizushima N (2007) Autophagy: process and function. Genes Dev 21:2861-2873
124
136. Mizushima N, Levine B,Cuervo AM, Klionsky DJ (2008) Autophagy fights disease
through cellular self-digestion. Nature 451:1069-1075
137. Moretti-Rojas I, Fuqua SA, Montgomery RA, Ill, McGuire WL (1988) A cDNA for
the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells. Breast
Cancer Res Treat 11:155-163
138. Morimoto H, Safrit JT, Bonavida B (1991) Synergistic effect of tumor necrosis
factor-alpha- and diphtheria toxin-mediated cytotoxicity in sensitive and resistant
human ovarian tumor cell lines. J Immunol 147:2609-2616
139. Morimoto H, Yonehara S, Bonavida B (1993) Overcoming tumor necrosis factor
and drug resistance of human tumor cell lines by combination treatment with antiFas antibody and drugs or toxins. Cancer Res 53:2591-2596
140. Morisset M, Capony F, Rochefort H (1986) Processing and estrogen regulation of
the 52-kilodalton protein inside MCF7 breast cancer cells. Endocrinology
119:2773-2782
141. Morissette M, Le SM, D'Astous M, Jourdain S, Al SS, Morin N, Estrada-Camarena
E, Mendez P, Garcia-Segura LM, Di PT (2008) Contribution of estrogen receptors
alpha and beta to the effects of estradiol in the brain. J Steroid Biochem Mol Biol
108:327-338
142. Narod SA, Foulkes WD (2004) BRCA1 and BRCA2: 1994 and beyond. Nat Rev
Cancer 4:665-676
125
143. Nash JD, Ozols RF, Smyth JF, Hamilton TC (1989) Estrogen and anti-estrogen
effects on the growth of human epithelial ovarian cancer in vitro. Obstet Gynecol
73:1009-1016
144. Newton K, Strasser A (1998) The Bcl-2 family and cell death regulation. Curr Opin
Genet Dev 8:68-75
145. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E,
Pettersson K, Warner M, Gustafsson JA (2001) Mechanisms of estrogen action.
Physiol Rev 81:1535-1565
146. Noy N (2007) Ligand specificity of nuclear hormone receptors: sifting through
promiscuity. Biochemistry 46:13461-13467
147. O'Connor PM, Kohn KW (1990) Comparative pharmacokinetics of DNA lesion
formation and removal following treatment of L1210 cells with nitrogen mustards.
Cancer Commun 2:387-394
148. O'Donnell AJ, Macleod KG, Burns DJ, Smyth JF, Langdon SP (2005) Estrogen
receptor-alpha mediates gene expression changes and growth response in ovarian
cancer cells exposed to estrogen. Endocr Relat Cancer 12:851-866
149. Olefsky JM (2001) Nuclear receptor minireview series. J Biol Chem 276:3686336864
150. Olive PL, Banath JP (1995) Sizing highly fragmented DNA in individual apoptotic
cells using the comet assay and a DNA crosslinking agent. Exp Cell Res 221:19-26
126
151. Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage
in individual cells. Nat Protoc 1:23-29
152. Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA
damage and repair in tumor and normal cells measured using the "comet" assay.
Radiat Res 122:86-94
153. Osborne CK, Yochmowitz MG, Knight WA, Ill, McGuire WL (1980) The value of
estrogen and progesterone receptors in the treatment of breast cancer. Cancer
46:2884-2888
154. Oxelmark E, Knoblauch R,Arnal S, Su LF, Schapira M, Garabedian MJ (2003)
Genetic dissection of p23, an Hsp90 cochaperone, reveals a distinct surface
involved in estrogen receptor signaling. J Biol Chem 278:36547-36555
155. Ozols RF (2005) Treatment goals in ovarian cancer. Int J Gynecol Cancer 15
Suppl 1:3-11
156. Ozols RF, Bundy BN, Greer BE, Fowler JM, Clarke-Pearson D, Burger RA, Mannel
RS, DeGeest K, Hartenbach EM, Baergen R (2003) Phase Ill trial of carboplatin
and paclitaxel compared with cisplatin and paclitaxel in patients with optimally
resected stage IlIl ovarian cancer: a Gynecologic Oncology Group study. J Clin
Oncol 21:3194-3200
157. Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, Domingo D,
Yahalom J (2001) A novel response of cancer cells to radiation involves autophagy
and formation of acidic vesicles. Cancer Res 61:439-444
127
158. Pal T, Permuth-Wey J, Betts JA, Krischer JP, Fiorica J, Arango H, LaPolla J,
Hoffman M, Martino MA, Wakeley K, Wilbanks G, Nicosia S, Cantor A, Sutphen R
(2005) BRCA1 and BRCA2 mutations account for a large proportion of ovarian
carcinoma cases. Cancer 104:2807-2816
159. Papac RJ (2001) Origins of cancer therapy. Yale J Biol Med 74:391-398
160. Parker MG (1991) Nuclear Hormone Receptors
161. Parker, Jensen EV (1991) Overview of the Nuclear Receptor Family.
162. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M,
Schneider MD, Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1dependent autophagy. Cell 122:927-939
163. Pearce ST, Jordan VC (2004) The biological role of estrogen receptors alpha and
beta in cancer. Crit Rev Oncol Hematol 50:3-22
164. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA (1997) Mouse estrogen
receptor beta forms estrogen response element-binding heterodimers with
estrogen receptor alpha. Mol Endocrinol 11:1486-1496
165. Pil PM, Lippard SJ (1992) Specific binding of chromosomal protein HMG1 to DNA
damaged by the anticancer drug cisplatin. Science 256:234-237
166. Pratt WB, Ruddon RW, Ensminger WD, Maybaum J (1994) The Anticancer drugs
167. Pujol P, Rey JM, Nirde P, Roger P, Gastaldi M, Laffargue F, Rochefort H,
Maudelonde T (1998) Differential expression of estrogen receptor-alpha and -beta
128
messenger RNAs as a potential marker of ovarian carcinogenesis. Cancer Res
58:5367-5373
168. Rao BR, Slotman BJ (1991) Endocrine factors in common epithelial ovarian cancer.
Endocr Rev 12:14-26
169. Rao GG, Miller DS (2005) Clinical applications of hormonal therapy in ovarian
cancer. Curr Treat Options Oncol 6:97-102
170. Razandi M, Pedram A, Merchenthaler I, Greene GL, Levin ER (2004) Plasma
membrane estrogen receptors exist and functions as dimers. Mol Endocrinol
18:2854-2865
171. Reardon JT, Vaisman A, Chaney SG, Sancar A (1999) Efficient nucleotide excision
repair of cisplatin, oxaliplatin, and Bis-aceto-ammine-dichloro-cyclohexylamineplatinum(IV) (JM216) platinum intrastrand DNA diadducts. Cancer Res 59:39683971
172. Reed (2006) Cisplatin, carboplatin and oxaliplatin. In: Chabner BA, Longo DL (eds)
Cancer chemotherapy and biotherapy, 4 edn
173. Reggiori F, Klionsky DJ (2002) Autophagy in the eukaryotic cell. Eukaryot Cell
1:11-21
174. Renan MJ (1993) How many mutations are required for tumorigenesis?
Implications from human cancer data. Mol Carcinog 7:139-146
129
175. Reynolds A, Anderson EM, Vermeulen A, Fedorov Y, Robinson K, Leake D,
Karpilow J, Marshall WS, Khvorova A (2006) Induction of the interferon response
by siRNA is cell type- and duplex length-dependent. RNA 12:988-993
176. Riggs BL, Khosla S, Melton U, 111(2002) Sex steroids and the construction and
conservation of the adult skeleton. Endocr Rev 23:279-302
177. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2001) Risk factors for epithelial borderline ovarian tumors: results of a
Swedish case-control study. Gynecol Oncol 83:575-585
178. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2002a) Risk factors for invasive epithelial ovarian cancer: results from
a Swedish case-control study. Am J Epidemiol 156:363-373
179. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Weiderpass E, Persson IR (2002b) Hormone replacement therapy and the risk of
invasive epithelial ovarian cancer in Swedish women. J Natl Cancer Inst 94:497504
180. Riman T, Dickman PW, Nilsson S, Nordlinder H, Magnusson CM, Persson IR
(2004) Some life-style factors and the risk of invasive epithelial ovarian cancer in
Swedish women. Eur J Epidemiol 19:1011-1019
181. Rink SM, Yarema KJ, Solomon MS, Paige LA, Tadayoni-Rebek BM, Essigmann
JM, Croy RG (1996) Synthesis and biological activity of DNA damaging agents that
form decoy binding sites for the estrogen receptor. Proc Natl Acad Sci U S A
93:15063-15068
130
182. Risch HA (2002) Hormone replacement therapy and the risk of ovarian cancer.
Gynecol Oncol 86:115-117
183. Roberts D, Schick J, Conway S, Biade S, Laub PB, Stevenson JP, Hamilton TC,
O'Dwyer PJ, Johnson SW (2005) Identification of genes associated with platinum
drug sensitivity and resistance in human ovarian cancer cells. Br J Cancer
92:1149-1158
184. Rochefort H (1990) Cathepsin D in breast cancer. Breast Cancer Res Treat 16:313
185. Rochefort H (1998) [Estrogens, cathepsin D and metastasis in cancers of the
breast and ovary: invasion or proliferation?]. C R Seances Soc Biol Fil 192:241-251
186. Rochefort H (1999) [Estrogen-induced genes in breast cancer, and their medical
importance]. Bull Acad Natl Med 183:955-968
187. Rose PG, Reale FR, Longcope C, Hunter RE (1990) Prognostic significance of
estrogen and progesterone receptors in epithelial ovarian cancer. Obstet Gynecol
76:258-263
188. Roy G, Horton JK, Roy R, Denning T, Mitra S, Boldogh 1(2000) Acquired alkylating
drug resistance of a human ovarian carcinoma cell line is unaffected by altered
levels of pro- and anti-apoptotic proteins. Oncogene 19:141-150
189. Russo IH,Russo J (1998) Role of hormones in mammary cancer initiation and
progression. J Mammary Gland Biol Neoplasia 3:49-61
131
190. Rutherford T, Brown WD, Sapi E, Aschkenazi S, Munoz A, Mor G (2000) Absence
of estrogen receptor-beta expression in metastatic ovarian cancer. Obstet Gynecol
96:417-421
191. Satokata 1,Tanaka K, Miura N, Narita M, Mimaki T, Satoh Y, Kondo S, Okada Y
(1992) Three nonsense mutations responsible for group A xeroderma
pigmentosum. Mutat Res 273:193-202
192. Saville B,Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA,
Safe S (2000) Ligand-, cell-, and estrogen receptor subtype (alpha/beta)dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:53795387
193. Sawyers C (2004) Targeted cancer therapy. Nature 432:294-297
194. Schaner ME, Ross DT, Ciaravino G, Sorlie T, Troyanskaya 0, Diehn M, Wang YC,
Duran GE, Sikic TL, Caldeira S, Skomedal H, Tu IP, Hernandez-Boussard T,
Johnson SW, O'Dwyer PJ, Fero MJ, Kristensen GB, Borresen-Dale AL, Hastie T,
Tibshirani R, van de RM, Teng NN, Longacre TA, Botstein D, Brown PO, Sikic BI
(2003) Gene expression patterns in ovarian carcinomas. Mol Biol Cell 14:43764386
195. Schildkraut JM, Schwingl PJ, Bastos E, Evanoff A, Hughes C (1996) Epithelial
ovarian cancer risk among women with polycystic ovary syndrome. Obstet Gynecol
88:554-559
196. Schuchard M, Landers JP, Sandhu NP, Spelsberg TC (1993) Steroid hormone
regulation of nuclear proto-oncogenes. Endocr Rev 14:659-669
132
197. Segnitz B, Gehring U (1995) Subunit structure of the nonactivated human estrogen
receptor. Proc Natl Acad Sci U S A 92:2179-2183
198. Segnitz B, Gehring U (1997) The function of steroid hormone receptors is inhibited
by the hsp90-specific compound geldanamycin. J Biol Chem 272:18694-18701
199. Sharma U, Marquis JC, Nicole DA, Hillier SM, Fedeles B, Rye PT, Essigmann JM,
Croy RG (2004) Design, synthesis, and evaluation of estradiol-linked genotoxicants
as anti-cancer agents. Bioorg Med Chem Lett 14:3829-3833
200. Shuey DJ, McCallus DE, Giordano T (2002) RNAi: gene-silencing in therapeutic
intervention. Drug Discov Today 7:1040-1046
201. Slotman BJ, Rao BR (1988) Ovarian cancer (review). Etiology, diagnosis,
prognosis, surgery, radiotherapy, chemotherapy and endocrine therapy. Anticancer
Res 8:417-434
202. States JC, McDuffie ER, Myrand SP, McDowell M, Cleaver JE (1998) Distribution
of mutations in the human xeroderma pigmentosum group A gene and their
relationships to the functional regions of the DNA damage recognition protein. Hum
Mutat 12:103-113
203. Stenoien DL, Mancini MG, Patel K,Allegretto EA, Smith CL, Mancini MA (2000)
Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1.
Mol Endocrinol 14:518-534
204. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, Mancini
MA (2001) FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and
proteasome-dependent. Nat Cell Biol 3:15-23
133
205. Stevens EV, Nishizuka S, Antony S, Reimers M, Varma S, Young L, Munson PJ,
Weinstein JN, Kohn EC, Pommier Y (2008) Predicting cisplatin and trabectedin
drug sensitivity in ovarian and colon cancers. Mol Cancer Ther 7:10-18
206. Sunters A, Springer CJ, Bagshawe KD, Souhami RL, Hartley JA (1992) The
cytotoxicity, DNA crosslinking ability and DNA sequence selectivity of the aniline
mustards melphalan, chlorambucil and 4-[bis(2-chloroethyl)amino] benzoic acid.
Biochem Pharmacol 44:59-64
207. Tew, Colvin OM, Jones RB (2006) Clinical and High-Dose Alkylating Agents. In:
Chabner BA, Longo DL (eds) Cancer Chemotherapy and Biotherapy, 4 edn
208. Treiber DK, Zhai X, Jantzen HM, Essigmann JM (1994) Cisplatin-DNA adducts are
molecular decoys for the ribosomal RNA transcription factor hUBF (human
upstream binding factor). Proc Natl Acad Sci U S A 91:5672-5676
209. Turteltaub KW, Dingley KH (1998) Application of accelerated mass spectrometry
(AMS) in DNA adduct quantification and identification. Toxicol Lett 102-103:435439
210. Turteltaub KW, Felton JS, Gledhill BL, Vogel JS, Southon JR, Caffee MW, Finkel
RC, Nelson DE, Proctor ID, Davis JC (1990) Accelerator mass spectrometry in
biomedical dosimetry: relationship between low-level exposure and covalent
binding of heterocyclic amine carcinogens to DNA. Proc Natl Acad Sci U S A
87:5288-5292
211. Tyagi A, Singh RP, Agarwal C, Siriwardana S, Sclafani RA, Agarwal R (2005)
Resveratrol causes Cdc2-tyrl5 phosphorylation via ATM/ATR-Chkl/2-Cdc25C
134
pathway as a central mechanism for S phase arrest in human ovarian carcinoma
Ovcar-3 cells. Carcinogenesis 26:1978-1987
212. Vandenabeele P, Vanden BT, Festjens N (2006) Caspase inhibitors promote
alternative cell death pathways. Sci STKE 2006:e44
213. Venkitaraman AR (2004) Tracing the network connecting BRCA and Fanconi
anaemia proteins. Nat Rev Cancer 4:266-276
214. von Angerer E, Prekajac J, Strohmeier J (1984) 2-Phenylindoles. Relationship
between structure, estrogen receptor affinity, and mammary tumor inhibiting
activity in the rat. J Med Chem 27:1439-1447
215. Walker G, MacLeod K, Williams AR, Cameron DA, Smyth JF, Langdon SP (2007)
Estrogen-regulated gene expression predicts response to endocrine therapy in
patients with ovarian cancer. Gynecol Oncol 106:461-468
216. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E,
Scrace G,Waterfield M, . (1985) Cloning of the human estrogen receptor cDNA.
Proc Natl Acad Sci U S A 82:7889-7893
217. Webb P, Lopez GN, Uht RM, Kushner PJ (1995) Tamoxifen activation of the
estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9:443-456
218. Webb P, Nguyen P,Valentine C, Lopez GN, Kwok GR, McInemey E,
Katzenellenbogen BS, Enmark E, Gustafsson JA, Nilsson S, Kushner PJ (1999)
The estrogen receptor enhances AP-1 activity by two distinct mechanisms with
135
different requirements for receptor transactivation functions. Mol Endocrinol
13:1672-1685
219. Wilson TM, Ewel A, Duguid JR, Eble JN, Lescoe MK, Fishel R, Kelley MR (1995)
Differential cellular expression of the human MSH2 repair enzyme in small and
large intestine. Cancer Res 55:5146-5150
220. Wu X, Fan W, Xu S, Zhou Y (2003) Sensitization to the cytotoxicity of cisplatin by
transfection with nucleotide excision repair gene xeroderma pigmentosun group A
antisense RNA in human lung adenocarcinoma cells. Clin Cancer Res 9:58745879
221. Xu Y, Traystman RJ, Hurn PD, Wang MM (2004) Membrane restraint of estrogen
receptor alpha enhances estrogen-dependent nuclear localization and genomic
function. Mol Endocrinol 18:86-96
222. Yap TA, Carden CP, Kaye SB (2009) Beyond chemotherapy: targeted therapies in
ovarian cancer. Nat Rev Cancer 9:167-181
223. Zakeri Z, Bursch W, Tenniswood M, Lockshin RA (1995) Cell death: programmed,
apoptosis, necrosis, or other? Cell Death Differ 2:87-96
224. Zang XP, Pento JT (2008) SiRNA inhibition of ER-alpha expression reduces KGFinduced proliferation of breast cancer cells. Anticancer Res 28:2733-2735
225. Zheng L,Annab LA, Afshari CA, Lee WH, Boyer TG (2001) BRCA1 mediates
ligand-independent transcriptional repression of the estrogen receptor. Proc Natl
Acad Sci U S A 98:9587-9592
136
226. Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in
perspective. Nature 408:433-439
137
138
i)
1(1
Chapter 3
Role of ER in E2-7a toxicity:
Studies in ER(+) and ER(-)
cell populations
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Introduction
Nitrogen mustards (mechlorethamine and tris(p-chloroethyl)amine) were the first nonhormonal agents to show significant antitumor activity in humans (Chabner and Roberts,
Jr., 2005). These agents have been a ubiquitous element of chemotherapy ever since,
and several modifications have been made to the parent mechlorethamine molecule to
develop a range of more effective therapeutics. Four of the nitrogen mustard derivatives
have been found to be superior to their parent compound - melphalan, chlorambucil,
cyclophosphamide and ifosfamide. They display a higher therapeutic index, broader
range of clinical activity and can be administered both orally and intravenously (Tew et
al., 2006). These alkylating agents have found their niche in the chemotherapeutic
domain, but the quest for an anticancer agent that is selective for tumor tissues and
spares normal tissues continues. Targeted therapy involves the development of a new
generation of chemotherapeutics meant to interfere specifically with a molecular target
that is believed to play a crucial role in tumor growth and progression (Hurley, 2002;
Sawyers, 2004). The vast increase of knowledge of the biological, genetic and molecular
aspects of neoplastic disease has resulted in more options to pursue. The Essigmann
lab is particularly interested in developing selective genotoxicants that interact with DNA;
we have leveraged the lessons learned from the study of the mechanisms of action of
DNA damaging drugs such as cisplatin in order to develop better, more selective antitumor agents.
One of the cisplatin mechanisms suggests that the combination of a DNA damaging
functionality with a ligand for a tumor-specific protein provides a novel way to improve
the therapeutic index of antitumor agents. In this regard, steroid hormones and their
receptors make attractive targets for drug development. Hormone-receptor interactions
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play a crucial role in the development and progression of many hormonally-responsive
cancers including those of the prostate, breast and ovary. We have synthesized a series
of such agents that are designed to introduce DNA damage in cancer cells while sparing
non-target tissues. The premise behind the design is that selectivity can be conferred by
combining a steroid receptor recognition moiety and a bi-functional DNA damaging
group (Figure 1.3). Our lab has previously reported the synthesis of an agent designed
to target estrogen receptor (ER) positive cancers (Mitra et al., 2002). This agent,
estradiol-C6NC2-N,N-bis-(2-chloroethyl)-aniline (E2-7a), contains a DNA damaging
aniline mustard moiety tethered to the estradiol through a stable linker.
The hypothesis for the mechanism of action of E2-7a is that the drug-DNA adduct
interacts with the ER through the estradiol moiety of E2-7a, thereby mediating cell death
selectively in cancer cells through one or more of the following routes: (i) Because they
are shielded by the ER, the DNA adducts formed by E2-7a are difficult for ER(+) tumor
cells to repair; the adducts therefore persist, and selectively disrupt growth and survival
mechanisms in ER(+) tumors, thereby sensitizing cells to the cytotoxic effects of the drug.
(ii) Drug-DNA adducts sequester the ER away from the protein's site of transcriptional
activity, and thereby disrupt the transcription of downstream ER-regulated genes
(transcription factor hijacking) (Rink et al., 1996). In other words, formation of the
receptor-adduct complex antagonizes the expression of downstream ER-responsive
genes and causes the disruption of cellular processes in ER(+) cells, leading to cell
death.
We have detected a promising indication of the differential toxicity of E2-7a in ER(+) vs.
ER(-) breast cancer cell lines, as described in Mitra et al. (2002). There was significantly
increased sensitivity towards E2-7a in MCF-7 cells (ER(+)) compared to the ER(-) breast
141
2
~
cancer cell line MDA-MB-231. This difference was not observed when the two cell lines
were treated with chlorambucil. Chlorambucil contains the same DNA damaging moiety
as does E2-7a and is expected to form similar kinds of DNA damage. Therefore, it can
be deduced that the tethering of estradiol moiety to the aniline mustard might be
responsible for the selectivity towards E2-7a. This prompted further studies into the
mechanism of action of E2-7a in the breast cancer cell lines.
In order to understand the possible contribution of repair shielding to E2-7a toxicity,
Accelerator Mass Spectrometry (AMS) was employed by researchers in the Essigmann
and Tannenbaum laboratories. AMS can be used to measure very low levels of
radioactivity associated with DNA; its sensitivity is almost five orders of magnitude
greater than conventional decay counting techniques (Liberman et al., 2004). AMS
enables the quantification of drug-DNA adducts, and correlations can be drawn between
the levels of adducts formed in culture (for which we can rapidly measure biological
effects) and adduct levels in target tissues of animal models (Turteltaub et al., 1990;
Turteltaub and Dingley, 1998). Shawn Hillier and John Marquis (Essigmann laboratory;
unpublished results) employed AMS to determine the level of DNA adducts in E2-7atreated cells. Their experiments show that the removal of [14C]-E2-7a-DNA adducts is
much slower in the ER(+) breast cancer cell line MCF-7 compared to the ER(-) MDAMB-231 cells. Under the same conditions, there was no difference in the level of
[14C]-melphalan-DNA adducts in MCF-7 vs. MDA-MB-231 cells. These preliminary
results indicate a favorable role for ER in retaining the drug-DNA adducts in cells, and in
inducing selective toxicity in ER(+) cells. This experiment is a step in the right direction
towards studying the mechanism of toxicity of E2-7a but the results must be interpreted
with the caveat in mind that the breast cancer cell lines possess other biochemical
142
differences besides ER status that might determine the ultimate outcome of drug action.
It would be more definitive if we could perform the same study with cell lines that have
varying levels of ER but are otherwise isogenic, thereby diminishing the above
biochemical differences.
One of the main objectives of my research is to understand whether receptor-drug
interaction affects the toxicity of E2-7ax. This chapter focuses on testing a pivotal
mechanistic element of E2-7a action - the role of ER in E2-7a toxicity. Towards this end,
we needed a system of ER(+) and ER(-) cell lines that are otherwise isogenic. Our
strategy to create such a system was to use the potential of RNA interference (RNAi), by
the means of which a chosen ER(+) cell population could be converted transiently into
an ER(-) population by depleting the ER levels in the cells.
RNAi is a fundamental pathway in eukaryotic cells where a sequence-specific small
interfering RNA (siRNA) molecule targets and degrades its complementary mRNA
(Elbashir et al., 2001). Naturally occurring siRNA molecules (21-23 nucleotides long) are
produced in cells by the cleavage of long pieces of double-stranded RNA by the enzyme
Dicer (Bernstein et al., 2001). The siRNA-Dicer complex recruits additional components
to form an RNA-induced silencing complex (RISC), which then targets a specific
complementary mRNA strand for enzymatic degradation, leading to post-transcriptional
gene silencing (Shuey et al., 2002). In addition to being produced in vivo, siRNA
molecules can also be synthetically produced, and introduced into cells to knockdown
the gene of choice. Synthetic siRNA molecules have an advantage over long doublestranded RNA molecules in that introducing long double-stranded RNA into cells results
in the interaction between the RNA molecules and intracellular RNA receptors, leading
to immune interferon response and shutdown of cellular protein production in target cells
143
(Reynolds et al., 2006). The siRNA molecules, on the other hand, are too short to cause
an interferon response. RNA interference pathway has been employed extensively to
study gene functions and the physiological effect of a gene product (Kim and Rossi,
2007; Kurreck, 2009).
Commercially available validated siRNA duplexes can be used to knockdown the gene
product of interest - ER, in our case. The estrogen receptor is a central protein in the
cellular network and is a transcription factor that regulates gene expression in response
to ligand binding (Nilsson et al., 2001; Pearce and Jordan, 2004). Therefore, knocking
down the ER could have several consequences on cell behavior - for instance, cell
growth might be affected since ER is involved in the regulation of proliferation. When the
receptor was knocked down in MCF-7 breast cancer cells, it significantly reduced cell
proliferation (Zang and Pento, 2008).
By knocking down the ER in target cells using RNAi, the role of the receptor in mediating
drug toxicity can be studied very effectively. At the same time, it is also desirable that the
growth profile of the ER-knocked down cells remain unaltered compared to the parental
cell line so that ER(-) cells are not inherently growth compromised even before drug
treatment. With these considerations in mind, the SKOV-3 cell line has been used for the
RNAi-based knockdown of ERa. These cells provide a model for ERa positive, cisplatin
resistant ovarian tumor, and more importantly, they are not growth responsive to
hormones in culture (Hua et al., 1995d), suggesting that titrating down the levels of ER
may not affect their growth profiles significantly.
SKOV-3 cells possess the ER, but do not respond to estrogens such as estradiol or
antiestrogens such as ICI 164,384. Although estrogen binding to ER occurs with a Kd
144
similar to that of canonical estrogen receptors such as those in MCF-7 cells (0.2 nM in
SKOV-3 cells compared to 0.4 nM in MCF-7; Coezy et al., 1982; Hua et al., 1995d) and
in various other systems (Kd ranging from 0.1-1nM; Kuiper et al., 1997), the ER present
in SKOV-3 seems to be deficient in the transcriptional regulation of selected growth
regulatory gene products. Hence, knocking down the ER would be expected to not affect
any of the growth characteristics of the cells. Thus, this cell line serves as an appropriate
model system to understand the interaction between ER and E2-7a without involving the
confounding effects of the transcriptional activity of the receptor. This system helps
address one of the two proposed mechanisms of action of E2-7a (repair shielding) and
understand the involvement of the ER in modulating E2-7a toxicity.
Studies describing the biological response of SKOV-3 cells to E2-7a were described in
detail in Chapter 2. We found that E2-7a was cytotoxic to SKOV-3 cells, and induced cell
death through autophagy. We also found evidence of DNA damage that might be
responsible for the persistent S-phase arrest observed with E2-7a treatment. In this
chapter, we extend our investigations of E2-7a to gain a more in-depth understanding of
the mechanism of action of E2-7a. The relative toxicity of E2-7a towards the parent
SKOV-3 cells has been tested and compared to the ER knockdown SKOV-3 cells. In
order to correlate the observed toxicity towards E2-7a with DNA adduct formation, drugDNA adduct levels have been measured in these distinct cell populations using AMS.
The results of these studies are described subsequently.
145
Materials and Methods
Chemicals:
Radiolabeled E2-7a (specific activity 20 mCi/mmol) used in these studies was
synthesized in our laboratory. Radiolabeled melphalan (specific activity 50 mCi/mmol)
was purchased from Moravek and re-purified using HPLC. Radiolabeled drugs were
diluted with unlabeled material where necessary. Cisplatin, melphalan, and 17p-estradiol
were obtained from Sigma-Aldrich. ICI 182,780 was a kind gift from Professor Robert
Hanson at Northeastern University. Antibodies to ER (sc-8002, mouse monoclonal) and
Actin (C-1 1, rabbit polyclonal) were obtained from Santa Cruz Biotechnology. Secondary
anti-mouse IgG and anti-rabbit IgG antibodies, both conjugated to Horseradish
Peroxidase, were obtained from Cell Signaling Technology (Beverly, MA).
Cell Culture:
SKOV-3 cells were purchased from the American Type Culture Collection (ATCC)
(Rockville, MD). Fetal Bovine Serum (FBS) and Charcoal-Dextran Treated FBS (CDTFBS) was obtained from Thermo Fisher Scientific (Logan, UT). SKOV-3 cells were
maintained in McCoy's 5a Medium (ATCC), supplemented with 10% FBS. Cells were
grown in a humidified 5%CO2/air atmosphere at 370 C.
For cell growth assays in the presence of estrogens/antiestrogens, cells were grown in
full medium for 24 hours, and transferred to phenol-red free medium with 10% CDT-FBS
146
and grown an additional 36 hours. E2-7c, melphalan, E2 and ICI 182,780 were
dissolved in DMSO and all treatments were carried out in phenol-red free medium with
CDT-FBS.
Transient ERa knockdown using siRNA:
Validated siRNA sequence from Stealth RNAi DuoPaks (ERa validated Stealth Select
RNAi, duplex 2; Invitrogen, Carlsbad, CA) was used to target ER. The Stealth RNAi
negative control (medium GC content; Invitrogen) was utilized as a control to ensure the
specificity of each targeted knockdown. Cells were plated at 40,000 per well in 6-well
plates, grown for 24 hours, followed by siRNA/NC treatment (25 nM) for 7 hours.
Lipofectamine 2000 (Invitrogen) was used as the transfection reagent, and the
procedure for transfection was as outlined in the Invitrogen product manual. Cells were
treated with test compounds one day after siRNA treatment. Gene knockdown was
verified using immunoblotting with antibody against ER.
For growth inhibition assays, cells were treated with the test compounds dissolved in
DMSO. Cells were detached using trypLE Express (Invitrogen) at times indicated and
the number of cells in control and drug-treated wells was determined using a Coulter
Counter. The percent growth inhibition was calculated as the ratio of cell number in
treated and control wells multiplied by 100. Significance was estimated by two-tailed Pvalues obtained from unpaired t-test calculated using GraphPad software with the mean
and standard deviation of growth inhibition value at each treatment point. Comparisons
with P<0.05 were considered to be significant.
147
Immunoblot Analysis:
Whole cell extracts were prepared by cell lysis using RIPA buffer (Santacruz; 1XTBS,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM sodium
orthovanadate, 1 mM NaF, 50 mM Tris-HCI (pH 7.4) and protease inhibitor cocktail
(Sigma-Aldrich)). Protein concentrations were determined using Bradford reagent with
BSA standard. Equal amounts of protein were separated on pre-cast 4-12% SDSpolyacrylamide gradient gels (Invitrogen) in MOPS buffer system and transferred to
Immobilon-P membranes (Millipore, Bedford, MA). Membranes were blocked with 5%
milk in Tris-Buffered Saline - Tween 20 (0.1% Tween 20, 10 mM Tris [pH 7.4], 150 mM
NaCl) and incubated with primary antibodies (against ERa or actin) overnight at 40C.
Membranes were incubated with secondary antibodies for 1-2 hours, and antibody
complexes formed with horseradish peroxidase-conjugated secondary antibodies were
visualized using Enhanced Chemiluminescence reagents (PicoWest, Pierce, Rockford,
IL).
Adduct measurement using Accelerator Mass Spectrometry:
SKOV-3 cells were exposed to various doses of [14C]-labeled test compounds (E2-7a or
melphalan). At the end of the incubation period, wells were washed once with fresh
medium, followed by a wash with PBS, and cells were detached from the plates using
trypLE express (Invitrogen), pelleted, and washed with phosphate buffered saline (PBS).
The cells were lysed in 0.5 mL of extraction buffer containing HEPES (50 mM), NaCl
(100 mM), EDTA (10 mM) and 0.8% SDS, followed by incubation with Proteinase K
(Roche Diagnostics; 0.6 mg/mL for 2 hours at 370C). After phenol-chloroform extraction
148
I I~
the aqueous phase was extracted with chloroform and the nucleic acids were
precipitated with 100% ethanol overnight at -200C, and subsequently pelleted by
centrifugation at 7000g for 15 min at 40C. The pellet was washed with 70% ethanol and
dried in vacuo. The dried pellet was reconstituted with 0.3 mL of the extraction buffer,
and incubated with RNase A (Roche Diagnostics; 0.9 mg/mL, 30 minutes at 37*C). DNA
was extracted by subsequent phenol:chloroform:isoamyl alcohol and chloroform:isoamyl
alcohol extractions. The aqueous phase from the second extraction was collected and
the DNA precipitated with 3 volumes of ice-cold ethanol, centrifuged and washed twice
with 70% ethanol. DNA was dissolved in 0.2 mL water and the concentration was
estimated by absorbance at 260 nm. All samples were adjusted to the same
concentration (4 pg/mL). AMS analyses were conducted at the Biological Engineering
Accelerator Mass Spectrometry (BEAMS) Lab at MIT by Paul Skipper and Rosa
Liberman as described in Liberman, et al. (2004). DNA solutions were applied directly to
a CuO matrix used for sample combustion. A standard consisting of a solution of [14Cmethyl] bovine serum albumin was used to calibrate the instrument. The amount of [14 C]
in each sample was calculated from the peak area ratio of the sample to the standard.
All samples were analyzed at least twice. Readings were obtained from AMS instrument
as dpm/pg DNA and converted to adducts per million bases.
Results
SKOV-3 cells are not growth sensitive to E2 or ICI 182,780. SKOV-3 cells were
grown in the presence of either the pure estrogen estradiol or the pure antiestrogens ICI
182,780 at various doses. The growth patterns are shown in Figure 3.1, and the
corresponding doubling times are shown in Table 3.1. There was no significant
149
difference in the cell numbers between the untreated cells growing in medium
supplemented with CDT-FBS and cells treated with various doses of estradiol or ICI
182,780. All the seven treatments resulted in similar doubling times, as can be seen in
Table 3.1. In addition, the cells grown in medium containing FBS grew at a similar rate
as the cells that were supplemented with CDT-FBS (data shown in Table 3.1). These
results indicate that as reported in the literature, SKOV-3 cells are not growth sensitive
to estradiol or pure antiestrogen in our system as well.
E2-7a is more toxic to the ER(+) SKOV-3 cells compared to ER(-) SKOV-3 cells.
Following treatment with siRNA against the ER or NC, cells were analyzed for their ER
levels by immunoblotting. Figure 3.2 shows a representative western blot of ER levels
with NC/siRNA treatments. It can be seen that 25 nM siRNA treatment effectively
reduced the ER levels by 92% within 24 hours of treatment (Figure 3.2). From the Figure,
it can be seen that the ER levels remained knocked down up to 72 hours after treatment
with siRNA (79% knockdown after 48 hours, and 90% knockdown after 72 hours). The
timeframe of subsequent ER knockdown experiments is well within 72 hours, and this
ensured that the levels of the receptor were low enough to create a transient ER(-)
population during the time course of all further experiments.
Figure 3.3 shows the results of treatment of the ER knockdown cells compared to the
NC-treated SKOV-3 cells with cisplatin and melphalan. As seen in Figure 3.3A, the
growth inhibition pattern of SKOV-3 ER(+) and ER(-) populations was identical following
cisplatin treatment, and the percent growth inhibition in the two populations was the
same at each dose in the range of doses from 1.25-20 pM. It appeared that knocking
down ER did not lead to a noticeable change in toxicity towards cisplatin. A similar
pattern was observed with melphalan, as can be seen from Figure 3.3B. There was no
150
significant difference in the toxicity of melphalan at various doses from 1.25-20 piM, and
as was the case with cisplatin, the ER status did not seem to affect the toxicity towards
melpahalan.
From Figure 3.4, we observe that E2-7a treatment, as opposed to cisplatin and
melphalan treatments, was able to induce a difference in cytotoxicity in ER(+) versus
ER(-) cells - there was a significant decrease in the toxicity of E2-7ca after ER
knockdown at 2.5, 5 and 10 gM doses. This difference in toxicity is not likely due to the
differences in growth rates of the ER(+) and ER(-) cells; preliminary experiments with
these populations showed that they grew with similar doubling times (ER(+) cells -39
hours and ER(-) cells-38 hours, data not shown). This clearly indicates a role for the
presence of the ER in SKOV-3 cells in inducing the additional toxicity observed in the
ER(+) cells compared to the ER knockdown cells.
E2-7a forms DNA-adducts in SKOV-3 cells. We wanted to examine whether there was
a correlation between the observed toxicity of E2-7a towards ER(+) and ER knockdown
SKOV-3 cells and the DNA-adduct levels in these populations. The first step of this study
was to test whether E2-7x-DNA adducts could be detected in these cells. This was done
by treating cells with [14C]-E2-7ax, isolating their DNA and measuring the radioactivity
associated with the DNA using AMS. The result of this experiment is shown in Figure 3.5.
We were able to detect E2-7a-DNA adducts at doses as low as 0.3 pM. The interesting
observation with the dose-response curve was that it was not a linear response. When
the dose was doubled from 0.31 to 0.62 pM, the adduct levels increased more than tenfold. Similarly, when the dose was doubled from 1.25 to 2.5 pM, the adduct level
151
increased from 14.8 to 68.0, demonstrating the non-linear nature of the dose-response
curve.
E2-7a adducts are higher in ER(+) vs. ER(-) treated SKOV-3 cells, whereas
melphalan treated cells show similar number of adducts in both cell populations.
A possible explanation for the observed differential toxicity of E2-7a between the ER(+)
and ER(-) populations is the repair shielding pathway by which E2-7a was designed. If
repair shielding were to occur in SKOV-3 cells, then the adduct levels would be expected
to be higher in those cells than in the SKOV-3 cells in which the siRNA had suppressed
the level of the ER. In order to address the question whether the presence of the ER
affected adduct levels, we exposed siRNA-treated and NC-treated SKOV-3 cells to
[14C]-E2-7c and measured the DNA-adduct levels after 8, 24 and 36 hours of drug
exposure. The results are shown in Figure 3.6A. Various relevant parameters associated
with the treatment, such as adduct levels, cell number and drug intake into cells, are
listed in Table 3.2. We observed that the adduct levels were significantly higher in the
ER(+) SKOV-3 cells compared to the siRNA-treated population (P <0.05 at all treatment
durations). Following an eight hour treatment, there were twice as many adducts in the
ER(+) as in the ER(-) cells. When the treatment duration was increased to 24 hours, the
ER(+) cells had almost four times as many adducts as the ER(-). A similar trend was
observed at 36 hours as well. This difference in adduct levels was not likely due to
decreased accumulation or transport of E2-7a into ER(-) cells, since we determined that
the amount of drug accumulating in the cells was the same in the ER(+) versus the ER(-)
populations (data is shown in Table 3.1; this finding was based on scintillation counting
of the distribution of the radioactive drug in the cells and medium). In fact, at all the
durations of treatment, both ER(+) and ER(-) had similar amounts of radioactivity
associated with the medium and with the cells. This indicated that (i) there was no
152
differential retention of the drug in the cells in the ER(+) compared to the ER(-) cells, and
(ii) there was an equilibrium between the amounts of drug in the cells and in the medium
over the course of treatment and furthermore, increasing the duration of treatment did
not lead to increased accumulation of the drug in the cells. In contrast to E2-7a,
melphalan produced a similar adduct burden in ER(+) and ER(-) cells, as can be seen in
Figure 3.6B. In addition, there was no change in the adduct levels over time of treatment,
and the adduct levels were lower than those observed with E2-7a. The experiment with
melphalan helps rule out artifacts in the siRNA treatment that might have caused the
differential sensitivity to E2-7a.
Discussion
DNA alkylating agents are very effective in killing cells that proliferate rapidly, and
consequently have demonstrated high efficacy as chemotherapeutics in the treatment of
potentially fatal tumors (Helleday et al., 2008; Hurley, 2002). One approach to improving
the therapeutic index of conventional alkylating agents is to target DNA repair machinery
selectively in cancer cells. We have chosen this route to develop a compound E2-7a that
alkylates DNA and contains a tumor-specific protein recognition moiety. The synthesis
and design strategy of this compound has been described in detail elsewhere (Mitra et
al., 2002; Sharma et al., 2004). E2-7a was designed to target cancers that aberrantly
express the estrogen receptor (ER), such as breast and ovarian cancers. This
compound was shown to be effective in inducing selective toxicity towards ER(+) cells
compared to the ER(-) breast cancer cells by Mitra et al (2002). The focus of this
dissertation is the application of E2-7a to the ovarian cancer scenario, and
understanding the mechanism of action of E2-7ca in ovarian cancer cells. Over 60% of
153
ovarian cancers express the ER (Brandenberger et al., 1998; Rao and Slotman, 1991)
and estrogen seems to play a key role in tumorigenesis of ovarian cancers (Clinton and
Hua, 1997; Langdon et al., 1994; Nash et al., 1989). Based on these pieces of evidence,
we hypothesized that E2-7a might be able to selectively induce toxicity in ER(+) ovarian
cancer cells compared to ER(-) cells. In this chapter, the mechanistic aspect of E2-7a
action has been probed further, and we have, in particular, tried to elucidate the role of
ER in the cytotoxicity of E2-7a towards ovarian cancer cells. A system of ER(+) and
ER(-) cell populations that are otherwise isogenic has been produced by treating the
parent ovarian cancer cell lines SKOV-3 with a negative control (NC) siRNA duplex and
an siRNA duplex against ER.
In order to confirm that the SKOV-3 cells were not growth-responsive to estrogens or
antiestrogens (it was necessary to obtain this information before we proceeded further
with the ER knockdown experiment), cells were grown in the presence of estradiol or the
pure antiestrogen ICI 182,780. It was found that the cells did not show any changes in
their growth rates in the presence of either ligand. Doses in the range of 1 nM - 100 nM
were tested, and there were no apparent differences in growth rates or cellular
morphology between control and treated cells.
This result is in agreement with the work done by Hua et al. (1995d) where the authors
had identified SKOV-3 cells to be an ovarian cancer cell line resistant to the effects of
estrogens and antiestrogens.
In their experiments, SKOV-3 cells were depleted of
steroids, and then treated with 0.1, 1 and 10 nM estradiol. It was found that the cell line
did not show any mitogenic effects in response to the estrogens. In addition, cells were
also treated with antiestrogens ICI 164,384 (pure antiestrogen) and 40H-tamoxifen
(partial antagonist) at various doses, and neither of these agents was able to induce any
154
changes in growth in these cells. Our experiments in this chapter confirmed the literature
findings; these results ensure that the cell line we are working with is not growth
sensitive to estrogens, and therefore it is likely that these cells will also not be growth
sensitive to the knockdown of ER.
In the experimental system employed in this chapter, specially designed and validated
siRNA duplexes against the ER were chosen and their concentrations were titrated to
confer the optimal level of knockdown. The knockdown of the receptor levels after siRNA
treatment was confirmed by immunoblotting experiments. It was determined that the ER
levels remained knocked down by 90% even at 72 hours after treatment with the siRNA
duplex (Figure 3.2), which ensured that during the time-frame of our subsequent
experiments with these cells, the ER levels remain suppressed.
The ER knockdown cells (ER(-)) and the ER(+) SKOV-3 cells were treated with E2-7a
and our results in Figure 3.4 clearly indicate that the absence of the ER makes the cells
less sensitive to the drug. This decreased sensitivity is unlikely to be due to any inherent
changes in growth patterns following ER knockdown; control experiments that measured
cell doubling time after the ER siRNA/negative control (NC) treatments confirmed that
the two cell populations grew at similar rates (data not shown). Therefore, the change in
growth inhibition patterns between ER(+) and ER(-) cells following E2-7aC treatment is
most likely a direct contribution of ER itself.
Although knocking down the ER in SKOV-3 did not affect the growth of the cells, we had
considered the possibility that the ER in SKOV-3 cells might be involved in determining
cell survival, which might in turn affect the sensitivity towards E2-7a. Several
experimental and clinical studies have implicated the ER in conferring neuroprotective
155
effects (Maggi et al., 2004; Morissette et al., 2008). Also, the ER has been shown to
induce the expression of anti-apoptotic bcl-2 gene expression in response to estradiol
through the interaction with cis-genomic Sp1 sites in MCF-7 and T47D breast cancer
cells (Dong et al., 1999). ER-mediated inhibition of NF-KB contributes to the antiinflammatory and protective effects of estrogen in breast cancer, bone health and
cardiovascular systems (Biswas et al., 2005; De et al., 2006). Based on these reports,
we considered it possible that knocking down the ER might render the SKOV-3 more
vulnerable to cytotoxic therapies, but the results from our control cytotoxicity
experiments with cisplatin and melphalan diminish the likelihood of this possibility. Both
cisplatin and melphalan treatments caused similar levels of growth inhibition in the ER(+)
and ER(-) cell populations. Also, the AMS experiment with [14C]-melphalan treatment
(Figure 3.6B) clearly demonstrated that a similar number of drug-DNA adducts were
being formed in the SKOV-3 ER(+) and ER(-) cells. These results reinforce the
conclusion that knocking down the ER did not make the cells inherently more sensitive
to cytotoxic agents and that the results we observed best fit with the hypothesis that ER
contributes to the change in the growth inhibition patterns between ER(+) and ER(-) cells
following E2-7a treatment. This result is strongly in favor of our intended mechanism of
action of E2-7a -- that is, the presence of the ER induces selective toxicity in ER(+) cells
compared to the ER knockdown cells.
We were interested in testing if the trend of drug-DNA adduct levels observed in each of
the populations aligned with the differential toxicity in order to investigate whether the
presence of the ER affected the adduct levels in treated cells. As mentioned earlier in
this chapter, there are at least two means by which interaction of the ER with drug-DNA
adduct can induce toxicity - repair shielding and transcription factor hijacking. In the
SKOV-3 system, the hijacking model is less likely to be operative as a factor in
156
differential toxicity because the ER in this cell line has been shown to be deficient in the
transcriptional activation of several of its canonical estrogen-responsive gene targets
such as progesterone receptor, HER2/neu and cathepsin D (Hua et al., 1995d).
In order to test whether the presence of the ER affected adduct levels, the DNA-adduct
levels were measured in SKOV-3 cell populations (ER(+) and ER(-)) following
continuous exposure to E2-7a. The results shown in Figure 3.6A indicate that the ER(+)
population endured a higher adduct burden compared to the ER(-) cells.
One difference between the ER(+) and ER(-) cells that could influence adduct levels is
the greater growth observed in the ER(-) cells during the treatment. It can be seen from
Table 3.2 that there were 1.3 times as many cells in the ER(-) compared to the ER(+)
population following the 36 hour treatment. Given this difference in cell number, we need
to ensure that the six-fold difference in adduct levels observed at this duration is still
meaningful. If drug-DNA adducts were continually being formed in both the populations,
then number of adducts per cell would still be six fold higher in the ER(+) compared to
the ER(-) cells, independent of the actual cell numbers in both the populations. If, on the
other hand, adduct formation were limited, then the existing adducts would be distributed
among the growing cells. In this case, the higher growth rate in the ER(-) cells can
account at the most for a 1.3 fold lower level of adducts per million bases. Therefore, in
either case, the increased adduct levels in the ER(+) population is an not artifact of
differential cell growth, and the significant six-fold increase in the ER(+) adduct levels is
a meaningful consequence of the presence of ER in these cells.
Melphalan treatment resulted in equivalent levels of adducts in both the cell populations.
In addition, it could be noticed that the adduct levels did not change with increasing
157
duration of treatment. This result likely indicates continual formation of adducts in both
the populations considering that the cell numbers are higher in the 36 hour time point
compared to the 8 hour time point. The melphalan cytotoxicity experiment (Figure 3.3B)
indicated that there was no difference in toxicity towards ER(+) and ER(-) cells. This
finding was consistent with the observation of similar adduct levels in this pair of cells
when treated with melphalan. Melphalan has the same DNA damaging moiety as E2-7a,
and is expected to form similar kind of DNA damage as E2-7a. Therefore, this
experiment makes it unlikely that inherent differences in the repair capacity of the ER(+)
and ER(-) cells might be responsible for the differential adduct levels. Since the cells are
expected to be isogenic except for their ER status, the result implies that the ER plays
an irrefutable role in increasing the adduct levels in E2-7a treated ER(+) cells above the
levels observed in the ER(-) population.
The increased toxicity of the ER(+) cells towards E2-7a is best explained by the role of
ER in facilitating the persistence of drug-DNA adducts in the ER(+) cells. The receptor
could mediate this effect either through its role in adduct shielding, or it could, through
some other means, enable increased formation of adducts in the ER(+) cells. The adduct
levels observed are the net effect of adduct formation and repair, and with the
continuous exposure to drug, it is not possible deconvolve the contribution of one from
the other with these studies. Nevertheless, the contribution of repair shielding to E2-7aL
toxicity is strongly supported by the experiments described in this chapter - ER prevents
the adducts from getting repaired, thereby resulting in net increased levels of adducts.
The increased adduct levels correlate well with the enhanced toxicity towards the drug in
ER(+) cells.
158
Conclusions
The design of novel targeted chemotherapeutics is indispensable in order to tackle the
issues of off-target effects and the emergence of resistance with existing therapies. We
have rationally designed a multifunctional genotoxicant that has the ability to overcome
both these concerns. By combining the ability to form adducts within DNA and to disrupt
cellular signaling into one agent, we have developed the compound E2-7aL that is
capable of selectively targeting cancer cells - E2-7a combines the modern targeted
approach with the traditional method of interfering with DNA replication.
In this chapter, the mechanistic basis of E2-7a toxicity was explored further, and we
found that the compound was selectively toxic to the ER(+) cells compared to the ER
knockdown SKOV-3 cells. In addition, these results were supported by the observation
that the E2-7a-DNA adduct levels were higher in the ER(+) compared to the ER(-) cell
population. Since the two cell populations are expected to be isogenic except for their
ER status, the observed difference in adduct levels is likely a direct contribution of the
ER. There was no differential toxicity observed in the ER(+) and ER knockdown cells
towards other alkylating agents such as melphalan and cisplatin. In addition, the drugDNA adduct levels in melphalan-treated cells were similar regardless of the ER status of
the cell population, indicating that the differential adducts levels observed with E2-7a
treatment was likely due to the interaction of the ER with the either E2-7a or the drugDNA complex or both. The results presented in this chapter strongly support the repair
shielding mechanism of E2-7a action and present a compelling case for the potential of
E2-7a as a targeted chemotherapeutic agent.
159
Future Directions
The adduct level measurements described in this chapter strongly support a role for the
ER in persistence of adducts in the ER(+) cells. Approaching the hypothesis from
another angle would make the results described in this chapter more substantial. We
would like to measure and characterize adduct levels in a more direct manner, and
towards this end, we are exploring the application of Triple Quadrupole Mass
Spectrometry (TQMS) in order detect specific drug-DNA base adducts and crosslinks.
This technique has shown promising results in detecting guanine adducts and guanineguanine crosslinks in preliminary in vitro experiments with E2-7a-treated calf-thymus
DNA. Once this technique is optimized to detect E2-7c-DNA adducts in SKOV-3 cells,
we would like to repeat the siRNA experiment and detect the adducts using the TQMS in
order to confirm the differential adduct levels in the ER(+) versus the ER(-) populations.
As mentioned in the previous section, the role of ER in creating more adducts in the
ER(+) cells is likely a net effect of the role of ER in repair shielding and in facilitating
increased rate of adduct formation. Once adducts are allowed to form, the rate of
removal of adducts could be determined by measuring adduct levels at the end of a
recovery period following drug treatment. Such experiments would help estimate the role
of ER in adduct repair and provide the basis for a more direct role for the ER in repair
shielding.
Another experiment that would support the repair shielding hypothesis is along similar
lines as the in vitro competition assay described by Mitra et al (2002). The authors had
added increasing amounts of estradiol to verify the gel shift caused by the E2-7a-DNA-
160
ER complex, and found that estradiol could diminish the gel shift by competitively
binding to the ER. We would like to perform a similar experiment by adding estradiol to
the SKOV-3 cells (ER(+) and the knockdown ER(-) cells) following E2-7a treatment and
observe whether the adduct levels are diminished in the ER(+) cells. This result would
test the specificity of drug-DNA adduct interaction with the ER, and if we do observe
diminished adduct levels in ER(+) cells, it would underscore the role of ER in repair
shielding. The experiments described above would further confirm the results described
in this chapter and provide additional evidence to reinforce the mechanism of action of
E2-7a.
161
Doubling times of SKOV-3 cells with estrogen
and antiestrogen treatment
Treatment
Doubling
time in
hours
Untreated (FBS)
30
Untreated(CDT-FBS)
30
E210nM
31
E2100nM
31
E21pM
30
ICI 10 nM
31
ICI 100 nM
32
ICI 1 pM
31
Table 3.1: Doubling times of SKOV-3 cells. All cells
(except untreated-FBS) were grown in phenol-red free
medium + CDT-FBS, and treated with estradiol (E2) or ICI
182,780 at various doses. The growth curves were fitted to
an exponential curve, and the doubling times were
calculated from the equation:
Doubling Time=ln(2)/kd
where kd is the growth constant inthe fitted exponential
curve N= Noexp(kd.t). N, No are cell numbers at time t and
to (time in hours). The data in the table is derived from the
growth curves in Figure 3.1
162
Adducts, cell numbers and ["4C]-E2-7a
distributions in ER(+) and ER(-) SKOV-3 cells
ER
ER
status
ER(+)
ER(-)
8h
24h
36h
8h
24h
36h
bases
43.8
91.2
80.0
22.9
30.5
12.9
Average
cell
number
(x 10-5)
1.02
1.49
1.54
1.07
1.77
2.02
Countscells
(x 10-4)
2.71
2.65
2.96
2.54
2.72
2.87
[14C]
Countsmedium
(x 10-4)
11.6
11.8
12.0
11.4
11.8
12.1
Adducts
per 106
[14C]
Table 3.2: Adducts, cell numbers and [14C] counts in
ER(+) and ER(-) cells. Cells were treated with 2 pM
[14C]-E2-7a for 8, 24 or 26 hours. Adducts per million
bases, cell numbers, radioactivity per well (cpm)
associated with cells and with the medium are shown for
each duration of treatment. The adduct levels vs. duration
of treatment is also shown in Figure 3.6.
163
-Chapter 3-
SKOV-3 cells are not growth-sensitive to
estradiol or ICI 182,780
Growth Characteristics - SKOV-3 cells
7006000500x
400 .a
E 300C
i200100
00
2
3
Time in days
LI Untreated ME2 1OnM
ElCllOnM
4
5
! E2 1OOnM E E2 1uM
ICl1OOnM MICIl1uM
Figure 3.1: SKOV-3 cells treated with various doses of 17pestradiol or pure antiestrogen ICI 182,780. The growth
pattern of the cells appears to be not affected by the
presence of estradiol or antiestrogen at various doses (Error
bars represent the standard deviation). The doubling time of
the cells with each of the treatments is listed in Table 3.1.
164
ER is downregulated after siRNA treatment for
up to 72 hours
NC
siRNA
72 hours
48 hours
24 hours
NC
siRNA
NC
siRNA
ER
(66 kDa)
-MEO
Actin
MO
-
Duration after
siRNA
treatment
Percentage
knockdown of
ER
24 hours
92.0
48 hours
78.9
72 hours
89.9
Figure 3.2: ER levels after knockdown using siRNA.
SKOV-3 cells treated with 25 nM siRNA against ER or
negative control (NC) for 7 hours, followed by incubation in
fresh medium for 24, 48 or 72 hours. Percentage
knockdown is relative to corresponding negative control
treatment and is determined by spot densitometry after
normalizing the actin levels. ER levels can be seen to be
knocked down up to 72 hours after siRNA treatment.
165
ER(+) and ER(-) SKOV-3 cells are equally
sensitive to cisplatin; they are equally sensitive
to melphalan as well
Cisplatin:
ER(+) vs. ER(-) SKOV-3 cells
100
80
60
40
20
0
0
5
15
10
Dose in uM
20
Melphalan:
ER(+) vs. ER(-) SKOV-3 cells
100
-c
.0-a
0
L~.
80
~1)
60
CU
C
'I)
0
40
(9
c,)
4-a
ci)
0~
20
0
5
15
10
Dose in uM
20
Figure 3.3: Cells treated with siRNA against ER or negative
control, followed by treatment for 36 hours with A. Cisplatin,
or B. Melphalan.There is no significant difference in the
response of the ER(+) vs. ER(-) populations to either of the
drugs.
166
ER(+) cells are more sensitive to E2-7a than ER(-)
cells
E2-7a:
ER(+) vs. ER(-) SKOV-3 cells
+ ER(-)
100
0
0)
--A- ER(+)
80
60
40
20
0
0
10
5
15
20
Dose inuM
Figure 3.4: Cells treated with siRNA against ER or negative
control for 7 hours. After 24 hours, ER(+) and ER(-)
populations were treated with various doses of E2-7a for 36
hours. Adherent cells were collected, and percent growth
relative to corresponding untreated control was measured
and plotted. Error bars represent standard deviations.
Suppression of the ER in SKOV-3 cells decreases sensitivity
to E2-7a. The treatment doses of 2.5, 5 and 10 pM show
significant difference in growth percentage between ER(+)
and ER(-) populations (P<0.05).
167
E2-7a forms DNA adducts in SKOV-3 cells
80
C,)
C
Dose Response:
SKOV-3 cells treated with E2-7a
68.0
70
60
0
.E
50
40
CO
-o
30
14.8
20
8.6
8
10
2.6
0.3
0.6
1.3
2.5
5.0
Dose inuM
Figure 3.5: Cells treated with various doses of
radiolabeled E2-7a for 4 hours, DNA isolated and
adduct levels measured using AMS. The values
above the bars are the mean number of adducts per
million bases for each dose. Error bars represent
standard deviation.
168
E2-7a adduct levels are higher in ER(+)
compared to ER(-) SKOV-3 cell populations
Adduct levels:
E2-7a treatment
40120
-
ER(+)
+
ER(-)
C-1000
A
-80
60
40
20
8
12
16
20
24
28
36
32
40
Duration of treatment (hours)
Adduct levels:
Melphalan treatment
0020
-A ER(+)
+ER(-)
C
0
B
E
010
0.
8
12
16
20
24
28
32
36
40
Duration of treatment (hours)
Figure 3.6: Adduct levels in siRNA vs. NC treated SKOV-3
cells after treatment with A. E2-7ax, or B. Melphalan. E2-7ax
treated cells show significantly higher adduct levels in
ER(+) compared to ER(-) cells (P<0.05). Melphalan
treated ER(+) and ER(-) cells show similar adduct levels.
169
Reference List
1.
Abend M (2003) Reasons to reconsider the significance of apoptosis for cancer
therapy. Int J Radiat Biol 79:927-941
2.
Agarwal R, Kaye SB (2003) Ovarian cancer: strategies for overcoming resistance
to chemotherapy. Nat Rev Cancer 3:502-516
3.
Ahn HJ, Kim YS, Kim JU, Han SM, Shin JW, Yang HO (2004) Mechanism of taxolinduced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem
91:1043-1052
4.
Ali S, Metzger D, Bornert JM, Chambon P (1993) Modulation of transcriptional
activation by ligand-dependent phosphorylation of the human oestrogen receptor
A/B region. EMBO J 12:1153-1160
5.
American Cancer Society (2008) Cancer Facts and Figures.
6.
Arai M, Utsunomiya J, Miki Y (2004) Familial breast and ovarian cancers. Int J Clin
Oncol 9:270-282
7.
Araujo SJ, Nigg EA, Wood RD (2001) Strong functional interactions of TFIIH with
XPC and XPG in human DNA nucleotide excision repair, without a preassembled
repairosome. Mol Cell Biol 21:2281-2291
170
8.
Argento M, Hoffman P, Gauchez AS (2008) Ovarian cancer detection and
treatment: current situation and future prospects. Anticancer Res 28:3135-3138
9.
Armitage P, Doll (1954) The age distribution of cancer and a multi-stage theory of
carcinogenesis. Br J Cancer 8:1-12
10. Asahina H, Kuraoka 1,Shirakawa M, Morita EH, Miura N, Miyamoto I, Ohtsuka E,
Okada Y, Tanaka K (1994) The XPA protein is a zinc metalloprotein with an ability
to recognize various kinds of DNA damage. Mutat Res 315:229-237
11.
Balcome S, Park S, Quirk Dorr DR, Hafner L, Phillips L,Tretyakova N (2004)
Adenine-containing DNA-DNA cross-links of antitumor nitrogen mustards. Chem
Res Toxicol 17:950-962
12.
Bandera CA, Ye B, Mok SC (2003) New technologies for the identification of
markers for early detection of ovarian cancer. Curr Opin Obstet Gynecol 15:51-55
13. Bardin A, Hoffmann P, Boulle N, Katsaros D, Vignon F, Pujol P, Lazennec G (2004)
Involvement of estrogen receptor beta in ovarian carcinogenesis. Cancer Res
64:5861-5869
14. Barth H, Hoffmann I, Klein S, Kaszkin M, Richards J, Kinzel V (1996) Role of
cdc25-C phosphatase in the immediate G2 delay induced by the exogenous
factors epidermal growth factor and phorbolester. J Cell Physiol 168:589-599
15. Bast RC, Jr., Klug TL, St JE, Jenison E, Niloff JM, Lazarus H, Berkowitz RS,
Leavitt T, Griffiths CT, Parker L,Zurawski VR, Jr., Knapp RC (1983) A
radioimmunoassay using a monoclonal antibody to monitor the course of epithelial
ovarian cancer. N Engl J Med 309:883-887
171
16.
Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate
ribonuclease in the initiation step of RNA interference. Nature 409:363-366
17.
Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a
specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3-14
18.
Bishop JM (1991) Molecular themes in oncogenesis. Cell 64:235-248
19.
Biswas DK, Singh S, Shi Q, Pardee AB, Iglehart JD (2005) Crossroads of estrogen
receptor and NF-kappaB signaling. Sci STKE 2005:e27
20.
Bonadonna G, Brusamolino E, Valagussa P, Rossi A, Brugnatelli L, Brambilla C,
De LM, Tancini G, Bajetta E, Musumeci R, Veronesi U (1976) Combination
chemotherapy as an adjuvant treatment in operable breast cancer. N Engl J Med
294:405-410
21.
Bookman MA, Greer BE, Ozols RF (2003) Optimal therapy of advanced ovarian
cancer: carboplatin and paclitaxel versus cisplatin and paclitaxel (GOG158) and an
update on GOG0182-ICON5. Int J Gynecol Cancer 13 Suppl 2:149-155
22.
Borini A, Rebellato E (2008) Focus on breast and ovarian cancer. Placenta 29
Suppl B:184-190
23.
Botti J, Djavaheri-Mergny M, Pilatte Y, Codogno P (2006) Autophagy signaling and
the cogwheels of cancer. Autophagy 2:67-73
24.
Boulikas T, Vougiouka M (2004) Recent clinical trials using cisplatin, carboplatin
and their combination chemotherapy drugs (review). Oncol Rep 11:559-595
172
I
25. Bowler J, Lilley TJ, Pittam JD, Wakeling AE (1989) Novel steroidal pure
antiestrogens. Steroids 54:71-99
26.
Brandenberger AW, Lebovic DI, Tee MK, Ryan IP,Tseng JF, Jaffe RB, Taylor RN
(1999) Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial
and endometriosis-derived stromal cells. Mol Hum Reprod 5:651-655
27. Brandenberger AW, Tee MK, Jaffe RB (1998) Estrogen receptor alpha (ER-alpha)
and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma
and ovarian cancer cell lines: down-regulation of ER-beta in neoplastic tissues. J
Clin Endocrinol Metab 83:1025-1028
28. Brookes, Lawley PD (1961) The reaction of mono- and di-functional alkylating
agents with nucleic acids. Biochem J 80:496-503
29. Brown JM, Attardi LD (2005) The role of apoptosis in cancer development and
treatment response. Nat Rev Cancer 5:231-237
30.
Brown SJ, Kellett PJ, Lippard SJ (1993) lxr1, a yeast protein that binds to
platinated DNA and confers sensitivity to cisplatin. Science 261:603-605
31.
Bruhn, Toney JH, Lippard SJ (1990) Biological processing of DNA modified by
platinum compounds., 38 edn
32. Bucourt R, Vignau M, Torelli V (1978) New biospecific adsorbents for the
purification of estradiol receptor. J Biol Chem 253:8221-8228
33. Burger RA (2007) Experience with bevacizumab in the management of epithelial
ovarian cancer. J Clin Oncol 25:2902-2908
173
34.
Chabner BA, Longo DL (2009) Cancer Chemotherapy and Biotherapy
35.
Chabner BA, Roberts TG, Jr. (2005) Timeline: Chemotherapy and the war on
cancer. Nat Rev Cancer 5:65-72
36.
Chatterjee M, Mohapatra S, lonan A, Bawa G, li-Fehmi R, Wang X, Nowak J, Ye B,
Nahhas FA, Lu K, Witkin SS, Fishman D, Munkarah A, Morris R, Levin NK, Shirley
NN, Tromp G, Abrams J, Draghici S, Tainsky MA (2006) Diagnostic markers of
ovarian cancer by high-throughput antigen cloning and detection on arrays. Cancer
Res 66:1181-1190
37.
Chen Z, Fadiel A, Jia JF, Sakamoto H, Carbone R, Naftolin F (1999) Induction of
apoptosis and suppression of clonogenicity of ovarian carcinoma cells with
estrogen mustard. Cancer 85:2616-2622
38.
Clark (2006) Molecular targeted drugs and growth factor receptor inhibitors. In:
Chabner BA, Longo DL (eds)
39.
Clarke PG (2002) Apoptosis: from morphological types of cell death to interacting
pathways. Trends Pharmacol Sci 23:308-309
40.
Clinton GM, Hua W (1997) Estrogen action in human ovarian cancer. Crit Rev
Oncol Hematol 25:1-9
41.
Coezy E, Borgna JL, Rochefort H (1982) Tamoxifen and metabolites in MCF7 cells:
correlation between binding to estrogen receptor and inhibition of cell growth.
Cancer Res 42:317-323
174
42.
Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS (1997) Tissue
distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and
estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and
ERalpha-knockout mouse. Endocrinology 138:4613-4621
43. Cowley SM, Hoare S, Mosselman S, Parker MG (1997) Estrogen receptors alpha
and beta form heterodimers on DNA. J Biol Chem 272:19858-19862
44. Cunat S, Hoffmann P, Pujol P (2004) Estrogens and epithelial ovarian cancer.
Gynecol Oncol 94:25-32
45. DaSilva JN, van Lier JE (1990) Synthesis and structure-affinity of a series of 7
alpha-undecylestradiol derivatives: a potential vector for therapy and imaging of
estrogen-receptor-positive cancers. J Med Chem 33:430-434
46. De BK, Vanden BW, Haegeman G (2006) Cross-talk between nuclear receptors
and nuclear factor kappaB. Oncogene 25:6868-6886
47.
Deng CX (2006) BRCA1: cell cycle checkpoint, genetic instability, DNA damage
response and cancer evolution. Nucleic Acids Res 34:1416-1426
48.
DeVita VT, Hellman S, Rosenberg SA (1997) Princples and Practice of Oncology,
5 edn
49.
Donahue BA, Augot M, Bellon SF, Treiber DK, Toney JH, Lippard SJ, Essigmann
JM (1990) Characterization of a DNA damage-recognition protein from mammalian
cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the
anticancer drug cisplatin. Biochemistry 29:5872-5880
175
50.
Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M, Samudio I, Kladde MP,
Vyhlidal C, Safe S (1999) Mechanisms of transcriptional activation of bcl-2 gene
expression by 17beta-estradiol in breast cancer cells. J Biol Chem 274:3209932107
51.
Driscoll MD, Sathya G, Muyan M, Klinge CM, Hilf R, Bambara RA (1998)
Sequence requirements for estrogen receptor binding to estrogen response
elements. J Biol Chem 273:29321-29330
52.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001)
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian
cells. Nature 411:494-498
53.
Essigmann JM, Rink SM, Park HJ, Croy RG (2001b) Design of DNA damaging
agents that hijack transcription factors and block DNA repair. Adv Exp Med Biol
500:301-313
54.
Essigmann, Rink SM, Park HJ, Croy RG (2001a) Design of DNA damaging agents
that hijack transcription factors and block DNA repair. In: Dansette (ed) Biological
Reactive Intermediates VI, 6 edn
55.
Festjens N, Vanden BT, Vandenabeele P (2006) Necrosis, a well-orchestrated
form of cell demise: signalling cascades, important mediators and concomitant
immune response. Biochim Biophys Acta 1757:1371-1387
56.
Fliss AE, Benzeno S, Rao J, Caplan AJ (2000) Control of estrogen receptor ligand
binding by Hsp90. J Steroid Biochem Mol Biol 72:223-230
176
57. Foulds (1954) The experimental study of tumor progression: a review. Cancer Res
14:327-339
58. Friedberg EC, Bonura T, Love JD, McMillan S, Radany EH, Schultz RA (1981) The
repair of DNA damage: recent developments and new insights. J Supramol Struct
Cell Biochem 16:91-103
59.
Friedberg EC, Walker G, Siede W (1995) DNA repair and mutagenesis
60.
Fu XD, Simoncini T (2008) Extra-nuclear signaling of estrogen receptors. IUBMB
Life 60:502-510
61.
Furuta T, Ueda T, Aune G, Sarasin A, Kraemer KH, Pommier Y (2002)
Transcription-coupled nucleotide excision repair as a determinant of cisplatin
sensitivity of human cells. Cancer Res 62:4899-4902
62. Gibb RK, Taylor DD, Wan T, O'Connor DM, Doering DL, Gercel-Taylor C (1997)
Apoptosis as a measure of chemosensitivity to cisplatin and taxol therapy in
ovarian cancer cell lines. Gynecol Oncol 65:13-22
63. Gozuacik D, Kimchi A (2007) Autophagy and cell death. Curr Top Dev Biol 78:217245
64. Grant DF, Bessho T, Reardon JT (1998) Nucleotide excision repair of melphalan
monoadducts. Cancer Res 58:5196-5200
65. Green S, Walter P, Greene G, Krust A, Goffin C, Jensen E, Scrace G,Waterfield M,
Chambon P (1986) Cloning of the human oestrogen receptor cDNA. J Steroid
Biochem 24:77-83
177
66. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J (1986) Sequence and
expression of human estrogen receptor complementary DNA. Science 231:11501154
67.
Greenlee RT, Murray T, Bolden S, Wingo PA (2000) Cancer statistics, 2000. CA
Cancer J Clin 50:7-33
68.
Haapala E, Hakala K, Jokipelto E, Vilpo J, Hovinen J (2001) Reactions of N,Nbis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'deoxyguanosine. Chem Res Toxicol 14:988-995
69. Hall JM, McDonnell DP (1999) The estrogen receptor beta-isoform (ERbeta) of the
human estrogen receptor modulates ERalpha transcriptional activity and is a key
regulator of the cellular response to estrogens and antiestrogens. Endocrinology
140:5566-5578
70.
Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, WhangPeng J, Rogan AM, Green WR, Ozols RF (1983) Characterization of a human
ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors.
Cancer Res 43:5379-5389
71.
Hamilton TC, Young RC, Ozols RF (1984) Experimental model systems of ovarian
cancer: applications to the design and evaluation of new treatment approaches.
Semin Oncol 11:285-298
72. Hanahan D,Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70
178
73. Hartley JA, Berardini MD, Souhami RL (1991) An agarose gel method for the
determination of DNA interstrand crosslinking applicable to the measurement of the
rate of total and "second-arm" crosslink reactions. Anal Biochem 193:131-134
74.
He Z, Ingles CJ (1997) Isolation of human complexes proficient in nucleotide
excision repair. Nucleic Acids Res 25:1136-1141
75. Helleday T, Petermann E, Lundin C, Hodgson B, Sharma RA (2008) DNA repair
pathways as targets for cancer therapy. Nat Rev Cancer 8:193-204
76. Hippert MM, O'Toole PS, Thorburn A (2006) Autophagy in cancer: good, bad, or
both? Cancer Res 66:9349-9351
77. Ho SM (2003) Estrogen, progesterone and epithelial ovarian cancer. Reprod Biol
Endocrinol 1:73
78.
Htun H, Holth LT, Walker D, Davie JR, Hager GL (1999) Direct visualization of the
human estrogen receptor alpha reveals a role for ligand in the nuclear distribution
of the receptor. Mol Biol Cell 10:471-486
79. Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995d) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
80.
Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995a) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
179
81.
Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995b) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
82.
Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995c) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
83. Huang A, Kaley G (2004) Gender-specific regulation of cardiovascular function:
estrogen as key player. Microcirculation 11:9-38
84. Huang JC, Zamble DB, Reardon JT, Lippard SJ, Sancar A (1994) HMG-domain
proteins specifically inhibit the repair of the major DNA adduct of the anticancer
drug cisplatin by human excision nuclease. Proc Natl Acad Sci U S A 91:1039410398
85. Hurley LH (2002) DNA and its associated processes as targets for cancer therapy.
Nat Rev Cancer 2:188-200
86. Igney FH, Krammer PH (2002) Death and anti-death: tumour resistance to
apoptosis. Nat Rev Cancer 2:277-288
87. Jacobs I, Bast RC, Jr. (1989) The CA 125 tumour-associated antigen: a review of
the literature. Hum Reprod 4:1-12
88. Jaffe N, Frei E, Ill, Traggis D, Bishop Y (1974) Adjuvant methotrexate and
citrovorum-factor treatment of osteogenic sarcoma. N Engl J Med 291:994-997
180
89.
Jakowlew SB, Breathnach R, Jeltsch JM, Masiakowski P, Chambon P (1984)
Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell
line MCF-7. Nucleic Acids Res 12:2861-2878
90.
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer
statistics, 2008. CA Cancer J Clin 58:71-96
91.
Jones CJ, Wood RD (1993) Preferential binding of the xeroderma pigmentosum
group A complementing protein to damaged DNA. Biochemistry 32:12096-12104
92.
Kim DH, Rossi JJ (2007) Strategies for silencing human disease using RNA
interference. Nat Rev Genet 8:173-184
93.
King WJ, Greene GL (1984) Monoclonal antibodies localize oestrogen receptor in
the nuclei of target cells. Nature 307:745-747
94.
Klein-Hitpass L, Ryffel GU, Heitlinger E, Cato AC (1988) A 13 bp palindrome is a
functional estrogen responsive element and interacts specifically with estrogen
receptor. Nucleic Acids Res 16:647-663
95.
Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M,
Baehrecke EH, Bahr BA, Ballabio A, Bamber BA, Bassham DC, Bergamini E, Bi X,
Biard-Piechaczyk M, Blum JS, Bredesen DE, Brodsky JL, Brumell JH, Brunk UT,
Bursch W, Camougrand N, Cebollero E, Cecconi F, Chen Y, Chin LS, Choi A, Chu
CT, Chung J, Clarke PG, Clark RS, Clarke SG, Clave C, Cleveland JL, Codogno P,
Colombo MI, Coto-Montes A, Cregg JM, Cuervo AM, Debnath J, Demarchi F,
Dennis PB, Dennis PA, Deretic V, Devenish RJ, Di SF, Dice JF, Difiglia M, neshKumar S, Distelhorst CW, Djavaheri-Mergny M, Dorsey FC, Droge W, Dron M,
181
Dunn WA, Jr., Duszenko M, Eissa NT, Elazar Z, Esclatine A, Eskelinen EL, Fesus
L, Finley KD, Fuentes JM, Fueyo J, Fujisaki K, Galliot B, Gao FB, Gewirtz DA,
Gibson SB, Gohla A, Goldberg AL, Gonzalez R, Gonzalez-Estevez C, Gorski S,
Gottlieb RA, Haussinger D, He YW, Heidenreich K, Hill JA, Hoyer-Hansen M, Hu X,
Huang WP, Iwasaki A, Jaattela M, Jackson WT, Jiang X, Jin S, Johansen T, Jung
JU, Kadowaki M, Kang C, Kelekar A, Kessel DH, Kiel JA, Kim HP, Kimchi A,
Kinsella TJ, Kiselyov K, Kitamoto K, Knecht E, Komatsu M, Kominami E, Kondo S,
Kovacs AL, Kroemer G, Kuan CY, Kumar R, Kundu M, Landry J, Laporte M, Le W,
Lei HY, Lenardo MJ, Levine B, Lieberman A, Lim KL, Lin FC, Liou W, Liu LF,
Lopez-Berestein G, Lopez-Otin C, Lu B, Macleod KF, Malorni W, Martinet W,
Matsuoka K, Mautner J, Meijer AJ, Melendez A, Michels P, Miotto G, Mistiaen WP,
Mizushima N, Mograbi B, Monastyrska 1,Moore MN, Moreira PI, Moriyasu Y, Motyl
T, Munz C, Murphy LO, Naqvi NI, Neufeld TP, Nishino I, Nixon RA, Noda T,
Nurnberg B, Ogawa M, Oleinick NL, Olsen LJ, Ozpolat B, Paglin S, Palmer GE,
Papassideri 1,Parkes M, Perlmutter DH, Perry G, Piacentini M, Pinkas-Kramarski
R, Prescott M, Proikas-Cezanne T, Raben N, Rami A, Reggiori F, Rohrer B,
Rubinsztein DC, Ryan KM, Sadoshima J, Sakagami H, Sakai Y, Sandri M,
Sasakawa C, Sass M, Schneider C, Seglen PO, Seleverstov 0, Settleman J,
Shacka JJ, Shapiro IM,Sibirny A, Silva-Zacarin EC, Simon HU, Simone C,
Simonsen A, Smith MA, Spanel-Borowski K, Srinivas V, Steeves M, Stenmark H,
Stromhaug PE, Subauste CS, Sugimoto S, Sulzer D, Suzuki T, Swanson MS,
Tabas I, Takeshita F, Talbot NJ, Talloczy Z, Tanaka K, Tanaka K, Tanida 1,Taylor
GS, Taylor JP, Terman A, Tettamanti G, Thompson CB, Thumm M, Tolkovsky AM,
Tooze SA, Truant R, Tumanovska LV, Uchiyama Y, Ueno T, Uzcategui NL, van dK,
I, Vaquero EC, Vellai T, Vogel MW, Wang HG, Webster P, Wiley JW, Xi Z, Xiao G,
Yahalom J, Yang JM, Yap G, Yin XM, Yoshimori T, Yu L, Yue Z, Yuzaki M,
182
Zabirnyk 0, Zheng X, Zhu X, Deter RL (2008) Guidelines for the use and
interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy
4:151-175
96. Koberle B, Roginskaya V, Wood RD (2006) XPA protein as a limiting factor for
nucleotide excision repair and UV sensitivity in human cells. DNA Repair (Amst)
5:641-648
97.
Kohn KW (1996) Beyond DNA cross-linking: history and prospects of DNAtargeted cancer treatment--fifteenth Bruce F. Cain Memorial Award Lecture.
Cancer Res 56:5533-5546
98. Kohn KW, Hartley JA, Mattes WB (1987) Mechanisms of DNA sequence selective
alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res
15:10531-10549
99. Krasner C (2007) Aromatase inhibitors in gynecologic cancers. J Steroid Biochem
Mol Biol 106:76-80
100. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson
JA (1997) Comparison of the ligand binding specificity and transcript tissue
distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870
101. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996) Cloning of
a novel receptor expressed in rat prostate and ovary. Proc Nati Acad Sci U S A
93:5925-5930
183
102. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van
der BB, Gustafsson JA (1998a) Interaction of estrogenic chemicals and
phytoestrogens with estrogen receptor beta. Endocrinology 139:4252-4263
103. Kuiper GG, Shughrue PJ, Merchenthaler I, Gustafsson JA (1998b) The estrogen
receptor beta subtype: a novel mediator of estrogen action in neuroendocrine
systems. Front Neuroendocrinol 19:253-286
104. Kumar R, Thompson EB (1999) The structure of the nuclear hormone receptors.
Steroids 64:310-319
105. Kundu GC, Schullek JR, Wilson IB (1994) The alkylating properties of chlorambucil.
Pharmacol Biochem Behav 49:621-624
106. Kurreck J (2009) RNA interference: from basic research to therapeutic applications.
Angew Chem Int Ed Engl 48:1378-1398
107. Kushner PJ, Agard D, Feng WJ, Lopez G, Schiau A, Uht R, Webb P, Greene G
(2000a) Oestrogen receptor function at classical and alternative response
elements. Novartis Found Symp 230:20-26
108. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P
(2000b) Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311317
109. Lahooti H, White R, Danielian PS, Parker MG (1994) Characterization of liganddependent phosphorylation of the estrogen receptor. Mol Endocrinol 8:182-188
184
110. Langdon SP, Hirst GL, Miller EP, Hawkins RA, Tesdale AL, Smyth JF, Miller WR
(1994) The regulation of growth and protein expression by estrogen in vitro: a
study of 8 human ovarian carcinoma cell lines. J Steroid Biochem Mol Biol 50:131135
111. Langdon SP, Ritchie A, Young K, Crew AJ, Sweeting V, Bramley T, Hillier S,
Hawkins RA, Tesdale AL, Smyth JF, . (1993) Contrasting effects of 17 betaestradiol on the growth of human ovarian carcinoma cells in vitro and in vivo. Int J
Cancer 55:459-464
112. Lau KM, Mok SC, Ho SM (1999) Expression of human estrogen receptor-alpha
and -beta, progesterone receptor, and androgen receptor mRNA in normal and
malignant ovarian epithelial cells. Proc Natl Acad Sci U S A 96:5722-5727
113. Lawley PD (1966) Effects of some chemical mutagens and carcinogens on nucleic
acids. Prog Nucleic Acid Res Mol Biol 5:89-131
114. Lazennec G (2006) Estrogen receptor beta, a possible tumor suppressor involved
in ovarian carcinogenesis. Cancer Lett 231:151-157
115. Le GP, Montano MM, Schodin DJ, Katzenellenbogen BS (1994) Phosphorylation of
the human estrogen receptor. Identification of hormone-regulated sites and
examination of their influence on transcriptional activity. J Biol Chem 269:44584466
116. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular
mechanisms and biological functions of autophagy. Dev Cell 6:463-477
185
117. Li M, HERTZ R, BERGENSTAL DM (1958) Therapy of choriocarcinoma and
related trophoblastic tumors with folic acid and purine antagonists. N Engl J Med
259:66-74
118. Liberman RG, Tannenbaum SR, Hughey BJ, Shefer RE, Klinkowstein RE, Prakash
C, Harriman SP, Skipper PL (2004) An interface for direct analysis of (14)c in
nonvolatile samples by accelerator mass spectrometry. Anal Chem 76:328-334
119. Lindahl T (1979) DNA glycosylases, endonucleases for apurinic/apyrimidinic sites,
and base excision-repair. Prog Nucleic Acid Res Mol Biol 22:135-192
120. Lindgren PR, Cajander S, Backstrom T, Gustafsson JA, Makela S, Olofsson JI
(2004) Estrogen and progesterone receptors in ovarian epithelial tumors. Mol Cell
Endocrinol 221:97-104
121. Lindor NM, Petersen GM, Hadley DW, Kinney AY, Miesfeldt S, Lu KH, Lynch P,
Burke W, Press N (2006) Recommendations for the care of individuals with an
inherited predisposition to Lynch syndrome: a systematic review. JAMA 296:15071517
122. Lukanova A, Kaaks R (2005) Endogenous hormones and ovarian cancer:
epidemiology and current hypotheses. Cancer Epidemiol Biomarkers Prev 14:98107
123. MacLeod MC, Powell KL, Tran N (1995) Binding of the transcription factor, Sp1, to
non-target sites in DNA modified by benzo[a]pyrene diol epoxide. Carcinogenesis
16:975-983
186
124. Maggi A, Ciana P, Belcredito S, Vegeto E (2004) Estrogens in the nervous system:
mechanisms and nonreproductive functions. Annu Rev Physiol 66:291-313
125. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K,
Blumberg B, Kastner P, Mark M, Chambon P, Evans RM (1995) The nuclear
receptor superfamily: the second decade. Cell 83:835-839
126. Masiakowski P, Breathnach R, Bloch J, Gannon F, Krust A, Chambon P (1982)
Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human
breast cancer cell line. Nucleic Acids Res 10:7895-7903
127. Masood S, Heitmann J, Nuss RC, Benrubi GI (1989) Clinical correlation of
hormone receptor status in epithelial ovarian cancer. Gynecol Oncol 34:57-60
128. May FE, Westley BR (1988) Identification and characterization of estrogenregulated RNAs in human breast cancer cells. J Biol Chem 263:12901-12908
129. May FE, Westley BR (1995) Estrogen regulated messenger RNAs in human breast
cancer cells. Biomed Pharmacother 49:400-414
130. McA'Nulty MM, Lippard SJ (1996) The HMG-domain protein lxr1 blocks excision
repair of cisplatin-DNA adducts in yeast. Mutat Res 362:75-86
131. McA'Nulty MM, Whitehead JP, Lippard SJ (1996) Binding of lxr1, a yeast HMGdomain protein, to cisplatin-DNA adducts in vitro and in vivo. Biochemistry
35:6089-6099
132. Meijer AJ, Codogno P (2006) Signalling and autophagy regulation in health, aging
and disease. Mol Aspects Med 27:411-425
187
133. Merajver SD, Pham TM, Caduff RF, Chen M, Poy EL, Cooney KA, Weber BL,
Collins FS, Johnston C, Frank TS (1995) Somatic mutations in the BRCA1 gene in
sporadic ovarian tumours. Nat Genet 9:439-443
134. Meyn, Murray D (1986) Cell cycle Effects of alkylating agents.
135. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu
Q, Cochran C, Bennett LM, Ding W, . (1994) A strong candidate for the breast and
ovarian cancer susceptibility gene BRCA1. Science 266:66-71
136. Mitra K, Marquis JC, Hillier SM, Rye PT, Zayas B, Lee AS, Essigmann JM, Croy
RG (2002) A rationally designed genotoxin that selectively destroys estrogen
receptor-positive breast cancer cells. J Am Chem Soc 124:1862-1863
137. Mizushima N (2007) Autophagy: process and function. Genes Dev 21:2861-2873
138. Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease
through cellular self-digestion. Nature 451:1069-1075
139. Moretti-Rojas I, Fuqua SA, Montgomery RA, 111,
McGuire WL (1988) A cDNA for
the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells. Breast
Cancer Res Treat 11:155-163
140. Morimoto H, Safrit JT, Bonavida B (1991) Synergistic effect of tumor necrosis
factor-alpha- and diphtheria toxin-mediated cytotoxicity in sensitive and resistant
human ovarian tumor cell lines. J Immunol 147:2609-2616
188
141. Morimoto H, Yonehara S, Bonavida B (1993) Overcoming tumor necrosis factor
and drug resistance of human tumor cell lines by combination treatment with antiFas antibody and drugs or toxins. Cancer Res 53:2591-2596
142. Morisset M, Capony F, Rochefort H (1986) Processing and estrogen regulation of
the 52-kilodalton protein inside MCF7 breast cancer cells. Endocrinology
119:2773-2782
143. Morissette M, Le SM, DAstous M, Jourdain S, Al SS, Morin N, Estrada-Camarena
E, Mendez P, Garcia-Segura LM, Di PT (2008) Contribution of estrogen receptors
alpha and beta to the effects of estradiol in the brain. J Steroid Biochem Mol Biol
108:327-338
144. Mutch DG (2002) Surgical management of ovarian cancer. Semin Oncol 29:3-8
145. Narod SA, Foulkes WD (2004) BRCA1 and BRCA2: 1994 and beyond. Nat Rev
Cancer 4:665-676
146. Nash JD, Ozols RF, Smyth JF, Hamilton TC (1989) Estrogen and anti-estrogen
effects on the growth of human epithelial ovarian cancer in vitro. Obstet Gynecol
73:1009-1016
147. Newton K, Strasser A (1998) The Bcl-2 family and cell death regulation. Curr Opin
Genet Dev 8:68-75
148. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J,Andersson G, Enmark E,
Pettersson K, Warner M, Gustafsson JA (2001) Mechanisms of estrogen action.
Physiol Rev 81:1535-1565
189
149. Noy N (2007) Ligand specificity of nuclear hormone receptors: sifting through
promiscuity. Biochemistry 46:13461-13467
150. O'Connor PM, Kohn KW (1990) Comparative pharmacokinetics of DNA lesion
formation and removal following treatment of L1210 cells with nitrogen mustards.
Cancer Commun 2:387-394
151. O'Donnell AJ, Macleod KG, Burns DJ, Smyth JF, Langdon SP (2005) Estrogen
receptor-alpha mediates gene expression changes and growth response in ovarian
cancer cells exposed to estrogen. Endocr Relat Cancer 12:851-866
152. Ogawa S, Inoue S, Watanabe T, Hiroi H,Orimo A, Hosoi T, Ouchi Y, Muramatsu M
(1998) The complete primary structure of human estrogen receptor beta (hER beta)
and its heterodimerization with ER alpha in vivo and in vitro. Biochem Biophys Res
Commun 243:122-126
153. Olefsky JM (2001) Nuclear receptor minireview series. J Biol Chem 276:3686336864
154. Olive PL, Banath JP (1995) Sizing highly fragmented DNA in individual apoptotic
cells using the comet assay and a DNA crosslinking agent. Exp Cell Res 221:19-26
155. Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage
in individual cells. Nat Protoc 1:23-29
156. Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA
damage and repair in tumor and normal cells measured using the "comet" assay.
Radiat Res 122:86-94
190
157. Osborne CK, Yochmowitz MG, Knight WA, Ill, McGuire WL (1980) The value of
estrogen and progesterone receptors in the treatment of breast cancer. Cancer
46:2884-2888
158. Oxelmark E, Knoblauch R,Arnal S, Su LF, Schapira M, Garabedian MJ (2003)
Genetic dissection of p23, an Hsp90 cochaperone, reveals a distinct surface
involved in estrogen receptor signaling. J Biol Chem 278:36547-36555
159. Ozols RF (2005) Treatment goals in ovarian cancer. Int J Gynecol Cancer 15
Suppl 1:3-11
160. Ozols RF, Bundy BN, Greer BE, Fowler JM, Clarke-Pearson D, Burger RA, Mannel
RS, DeGeest K, Hartenbach EM, Baergen R (2003) Phase Ill trial of carboplatin
and paclitaxel compared with cisplatin and paclitaxel in patients with optimally
resected stage Ill ovarian cancer: a Gynecologic Oncology Group study. J Clin
Oncol 21:3194-3200
161. Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, Domingo D,
Yahalom J (2001) A novel response of cancer cells to radiation involves autophagy
and formation of acidic vesicles. Cancer Res 61:439-444
162. Pal T, Permuth-Wey J, Betts JA, Krischer JP, Fiorica J, Arango H, LaPolla J,
Hoffman M, Martino MA, Wakeley K,Wilbanks G, Nicosia S, Cantor A, Sutphen R
(2005) BRCA1 and BRCA2 mutations account for a large proportion of ovarian
carcinoma cases. Cancer 104:2807-2816
163. Papac RJ (2001) Origins of cancer therapy. Yale J Biol Med 74:391-398
164. Parker MG (1991) Nuclear Hormone Receptors
191
165. Parker, Jensen EV (1991) Overview of the Nuclear Receptor Family.
166. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA
Cancer J Clin 55:74-108
167. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M,
Schneider MD, Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1dependent autophagy. Cell 122:927-939
168. Pearce ST, Jordan VC (2004) The biological role of estrogen receptors alpha and
beta in cancer. Crit Rev Oncol Hematol 50:3-22
169. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA (1997) Mouse estrogen
receptor beta forms estrogen response element-binding heterodimers with
estrogen receptor alpha. Mol Endocrinol 11:1486-1496
170. Pil PM, Lippard SJ (1992) Specific binding of chromosomal protein HMG1 to DNA
damaged by the anticancer drug cisplatin. Science 256:234-237
171. Pratt WB, Ruddon RW, Ensminger WD, Maybaum J (1994) The Anticancer drugs
172. Pujol P, Rey JM, Nirde P, Roger P, Gastaldi M, Laffargue F, Rochefort H,
Maudelonde T (1998) Differential expression of estrogen receptor-alpha and -beta
messenger RNAs as a potential marker of ovarian carcinogenesis. Cancer Res
58:5367-5373
173. Rao BR, Slotman BJ (1991) Endocrine factors in common epithelial ovarian cancer.
Endocr Rev 12:14-26
192
174. Rao GG, Miller DS (2005) Clinical applications of hormonal therapy in ovarian
cancer. Curr Treat Options Oncol 6:97-102
175. Razandi M, Pedram A, Merchenthaler I, Greene GL, Levin ER (2004) Plasma
membrane estrogen receptors exist and functions as dimers. Mol Endocrinol
18:2854-2865
176. Reardon JT, Vaisman A, Chaney SG, Sancar A (1999) Efficient nucleotide excision
repair of cisplatin, oxaliplatin, and Bis-aceto-ammine-dichloro-cyclohexylamineplatinum(IV) (JM216) platinum intrastrand DNA diadducts. Cancer Res 59:39683971
177. Reed (2006) Cisplatin, carboplatin and oxaliplatin. In: Chabner BA, Longo DL (eds)
Cancer chemotherapy and biotherapy, 4 edn
178. Reggiori F, Klionsky DJ (2002) Autophagy in the eukaryotic cell. Eukaryot Cell
1:11-21
179. Renan MJ (1993) How many mutations are required for tumorigenesis?
Implications from human cancer data. Mol Carcinog 7:139-146
180. Reynolds A, Anderson EM, Vermeulen A, Fedorov Y, Robinson K, Leake D,
Karpilow J, Marshall WS, Khvorova A (2006) Induction of the interferon response
by siRNA is cell type- and duplex length-dependent. RNA 12:988-993
(2002) Sex steroids and the construction and
181. Riggs BL, Khosla S, Melton U, 111
conservation of the adult skeleton. Endocr Rev 23:279-302
193
182. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2001) Risk factors for epithelial borderline ovarian tumors: results of a
Swedish case-control study. Gynecol Oncol 83:575-585
183. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2002a) Risk factors for invasive epithelial ovarian cancer: results from
a Swedish case-control study. Am J Epidemiol 156:363-373
184. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Weiderpass E, Persson IR (2002b) Hormone replacement therapy and the risk of
invasive epithelial ovarian cancer in Swedish women. J Natl Cancer Inst 94:497504
185. Riman T, Dickman PW, Nilsson S, Nordlinder H, Magnusson CM, Persson IR
(2004) Some life-style factors and the risk of invasive epithelial ovarian cancer in
Swedish women. Eur J Epidemiol 19:1011-1019
186. Rimer, Schildkraut JM (1997) Cancer Screening. In: DeVita VT, Hellman S,
Rosenberg SA (eds), Fifth edn pp 619-631
187. Rink SM, Yarema KJ, Solomon MS, Paige LA, Tadayoni-Rebek BM, Essigmann
JM, Croy RG (1996) Synthesis and biological activity of DNA damaging agents that
form decoy binding sites for the estrogen receptor. Proc Natl Acad Sci U S A
93:15063-15068
188. Risch HA (2002) Hormone replacement therapy and the risk of ovarian cancer.
Gynecol Oncol 86:115-117
194
189. Roberts D, Schick J,Conway S, Biade S, Laub PB, Stevenson JP, Hamilton TC,
O'Dwyer PJ, Johnson SW (2005) Identification of genes associated with platinum
drug sensitivity and resistance in human ovarian cancer cells. Br J Cancer
92:1149-1158
190. Rochefort H (1990) Cathepsin D in breast cancer. Breast Cancer Res Treat 16:313
191. Rochefort H (1998) [Estrogens, cathepsin D and metastasis in cancers of the
breast and ovary: invasion or proliferation?]. C R Seances Soc Biol Fil 192:241-251
192. Rochefort H (1999) [Estrogen-induced genes in breast cancer, and their medical
importance]. Bull Acad Natl Med 183:955-968
193. Rose PG, Reale FR, Longcope C, Hunter RE (1990) Prognostic significance of
estrogen and progesterone receptors in epithelial ovarian cancer. Obstet Gynecol
76:258-263
194. Roy G, Horton JK, Roy R, Denning T, Mitra S, Boldogh 1(2000) Acquired alkylating
drug resistance of a human ovarian carcinoma cell line is unaffected by altered
levels of pro- and anti-apoptotic proteins. Oncogene 19:141-150
195. Russo IH,Russo J (1998) Role of hormones in mammary cancer initiation and
progression. J Mammary Gland Biol Neoplasia 3:49-61
196. Rutherford T, Brown WD, Sapi E,Aschkenazi S, Munoz A, Mor G (2000) Absence
of estrogen receptor-beta expression in metastatic ovarian cancer. Obstet Gynecol
96:417-421
195
197. Satokata 1,Tanaka K, Miura N, Narita M, Mimaki T, Satoh Y, Kondo S, Okada Y
(1992) Three nonsense mutations responsible for group A xeroderma
pigmentosum. Mutat Res 273:193-202
198. Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA,
Safe S (2000) Ligand-, cell-, and estrogen receptor subtype (alpha/beta)dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:53795387
199. Sawyers C (2004) Targeted cancer therapy. Nature 432:294-297
200. Schaner ME, Ross DT, Ciaravino G, Sorlie T, Troyanskaya 0, Diehn M, Wang YC,
Duran GE, Sikic TL, Caldeira S, Skomedal H, Tu IP, Hernandez-Boussard T,
Johnson SW, O'Dwyer PJ, Fero MJ, Kristensen GB, Borresen-Dale AL, Hastie T,
Tibshirani R, van de RM, Teng NN, Longacre TA, Botstein D, Brown PO, Sikic BI
(2003) Gene expression patterns in ovarian carcinomas. Mol Biol Cell 14:43764386
201. Schildkraut JM, Schwingl PJ, Bastos E, Evanoff A, Hughes C (1996) Epithelial
ovarian cancer risk among women with polycystic ovary syndrome. Obstet Gynecol
88:554-559
202. Schuchard M, Landers JP, Sandhu NP, Spelsberg TC (1993) Steroid hormone
regulation of nuclear proto-oncogenes. Endocr Rev 14:659-669
203. Segnitz B, Gehring U (1995) Subunit structure of the nonactivated human estrogen
receptor. Proc Natl Acad Sci U S A 92:2179-2183
196
204. Segnitz B, Gehring U (1997) The function of steroid hormone receptors is inhibited
by the hsp90-specific compound geldanamycin. J Biol Chem 272:18694-18701
205. Sharma U, Marquis JC, Nicole DA, Hillier SM, Fedeles B, Rye PT, Essigmann JM,
Croy RG (2004) Design, synthesis, and evaluation of estradiol-linked genotoxicants
as anti-cancer agents. Bioorg Med Chem Lett 14:3829-3833
206. Shuey DJ, McCallus DE, Giordano T (2002) RNAi: gene-silencing in therapeutic
intervention. Drug Discov Today 7:1040-1046
207. Slotman BJ, Rao BR (1988) Ovarian cancer (review). Etiology, diagnosis,
prognosis, surgery, radiotherapy, chemotherapy and endocrine therapy. Anticancer
Res 8:417-434
208. States JC, McDuffie ER, Myrand SP, McDowell M, Cleaver JE (1998) Distribution
of mutations in the human xeroderma pigmentosum group A gene and their
relationships to the functional regions of the DNA damage recognition protein. Hum
Mutat 12:103-113
209. Stenoien DL, Mancini MG, Patel K, Allegretto EA, Smith CL, Mancini MA (2000)
Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1.
Mol Endocrinol 14:518-534
210. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, Mancini
MA (2001) FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and
proteasome-dependent. Nat Cell Biol 3:15-23
197
211. Stevens EV, Nishizuka S, Antony S, Reimers M, Varma S, Young L, Munson PJ,
Weinstein JN, Kohn EC, Pommier Y (2008) Predicting cisplatin and trabectedin
drug sensitivity in ovarian and colon cancers. Mol Cancer Ther 7:10-18
212. Sunters A, Springer CJ, Bagshawe KD, Souhami RL, Hartley JA (1992) The
cytotoxicity, DNA crosslinking ability and DNA sequence selectivity of the aniline
mustards melphalan, chlorambucil and 4-[bis(2-chloroethyl)amino] benzoic acid.
Biochem Pharmacol 44:59-64
213. Tew, Colvin OM, Jones RB (2006) Clinical and High-Dose Alkylating Agents. In:
Chabner BA, Longo DL (eds) Cancer Chemotherapy and Biotherapy, 4 edn
214. Treiber DK, Zhai X, Jantzen HM, Essigmann JM (1994) Cisplatin-DNA adducts are
molecular decoys for the ribosomal RNA transcription factor hUBF (human
upstream binding factor). Proc Natl Acad Sci U S A 91:5672-5676
215. Turteltaub KW, Dingley KH (1998) Application of accelerated mass spectrometry
(AMS) in DNA adduct quantification and identification. Toxicol Lett 102-103:435439
216. Turteltaub KW, Felton JS, Gledhill BL, Vogel JS, Southon JR, Caffee MW, Finkel
RC, Nelson DE, Proctor ID, Davis JC (1990) Accelerator mass spectrometry in
biomedical dosimetry: relationship between low-level exposure and covalent
binding of heterocyclic amine carcinogens to DNA. Proc Natl Acad Sci U S A
87:5288-5292
217. Tyagi A, Singh RP, Agarwal C, Siriwardana S, Sclafani RA, Agarwal R (2005)
Resveratrol causes Cdc2-tyrl5 phosphorylation via ATM/ATR-Chk1/2-Cdc25C
198
pathway as a central mechanism for S phase arrest in human ovarian carcinoma
Ovcar-3 cells. Carcinogenesis 26:1978-1987
218. Vandenabeele P,Vanden BT, Festjens N (2006) Caspase inhibitors promote
alternative cell death pathways. Sci STKE 2006:e44
219. Venkitaraman AR (2004) Tracing the network connecting BRCA and Fanconi
anaemia proteins. Nat Rev Cancer 4:266-276
220. von Angerer E, Prekajac J, Strohmeier J (1984) 2-Phenylindoles. Relationship
between structure, estrogen receptor affinity, and mammary tumor inhibiting
activity in the rat. J Med Chem 27:1439-1447
221. Walker G, MacLeod K, Williams AR, Cameron DA, Smyth JF, Langdon SP (2007)
Estrogen-regulated gene expression predicts response to endocrine therapy in
patients with ovarian cancer. Gynecol Oncol 106:461-468
222. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E,
Scrace G,Waterfield M, . (1985) Cloning of the human estrogen receptor cDNA.
Proc Natl Acad Sci U S A 82:7889-7893
223. Webb P, Lopez GN, Uht RM, Kushner PJ (1995) Tamoxifen activation of the
estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9:443-456
224. Webb P, Nguyen P, Valentine C, Lopez GN, Kwok GR, McInerney E,
Katzenellenbogen BS, Enmark E, Gustafsson JA, Nilsson S, Kushner PJ (1999)
The estrogen receptor enhances AP-1 activity by two distinct mechanisms with
199
different requirements for receptor transactivation functions. Mol Endocrinol
13:1672-1685
225. Wilson TM, Ewel A, Duguid JR, Eble JN, Lescoe MK, Fishel R, Kelley MR (1995)
Differential cellular expression of the human MSH2 repair enzyme in small and
large intestine. Cancer Res 55:5146-5150
226. Wu X, Fan W, Xu S, Zhou Y (2003) Sensitization to the cytotoxicity of cisplatin by
transfection with nucleotide excision repair gene xeroderma pigmentosun group A
antisense RNA in human lung adenocarcinoma cells. Clin Cancer Res 9:58745879
227. Xu Y, Traystman RJ, Hurn PD, Wang MM (2004) Membrane restraint of estrogen
receptor alpha enhances estrogen-dependent nuclear localization and genomic
function. Mol Endocrinol 18:86-96
228. Yap TA, Carden CP, Kaye SB (2009) Beyond chemotherapy: targeted therapies in
ovarian cancer. Nat Rev Cancer 9:167-181
229. Zakeri Z, Bursch W, Tenniswood M, Lockshin RA (1995) Cell death: programmed,
apoptosis, necrosis, or other? Cell Death Differ 2:87-96
230. Zang XP, Pento JT (2008) SiRNA inhibition of ER-alpha expression reduces KGFinduced proliferation of breast cancer cells. Anticancer Res 28:2733-2735
231. Zheng L,Annab LA, Afshari CA, Lee WH, Boyer TG (2001) BRCA1 mediates
ligand-independent transcriptional repression of the estrogen receptor. Proc Natl
Acad Sci U S A 98:9587-9592
200
232. Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in
perspective. Nature 408:433-439
201
Chapter 4
Investigation of cytotoxicity and
other biological properties of
phenylindole compounds in
ovarian cancer cells
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Introduction
Ovarian cancer (OC) is the leading cause of gynecological deaths in the United States,
with 22,000 new cases and 15,000 deaths associated with the disease every year
(American Cancer Society, 2008). The five-year survival rate of OC disease ranges from
30 to 92%, depending on the severity of the spread of the disease at the time of
diagnosis (Jemal et al., 2008). The relative lack of specific early signs and symptoms of
OC combined with inefficient screening techniques results in overall low cure rates for
the disease. Currently, the treatment options for both primary and recurrent cancers are
limited, and research focuses on developing new modalities of selectively targeting OC
and its metastases. Debulking surgery remains key in OC treatment (Agarwal and Kaye,
2003); adjuvant chemotherapy has also been shown to be beneficial. Results from
randomized trails have now established platinum-taxol combination regimen as the firstline treatment for advanced OC (du Bois et al., 1999; Neijt et al., 2000; Ozols et al., 2003;
Piccart et al., 2000). Although the response rate to such therapies has been promising,
studies point to the outcome that most of the patients will relapse with a median
progression-free survival of 18 months (Greenlee et al., 2001). In this scenario, therapy
based on better understanding of the disease seems to be the most effective means to a
better prognosis for the patient (Yap et al., 2009).
Although the etiological origins of OC have not been fully identified, several studies point
to the role of estrogen in promoting tumorigenesis (Greenlee et al., 2001; Lukanova and
Kaaks, 2005). Estrogen has been shown to induce proliferation in many ER(+) cancer
cells, and antiestrogens have been reported to inhibit the mitogenic effect of estrogen in
ER(+) cells (Langdon et al., 1994; Nash et al., 1989). Epidemiological studies have
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found that hormonal replacement therapy can increase the risk of OC in
postmenopausal women, while oral contraceptive use has been associated with
decreased risk of developing OC (Cunat et al., 2004; Risch, 2002; Slotman and Rao,
1988). In addition, interventions with antiestrogens and aromatase inhibitors have
displayed some clinical benefits for OC patients (Cunat et al., 2005; Krasner, 2007;
Walker et al., 2007). These findings reinforce the crucial role played by estrogens and
their cognate receptors in the development and progression of OC. In this context,
estrogen receptor (ER), the mediator of estrogen action in ovarian systems, is an
attractive target for the development of drugs to treat primary and recurrent OC. ER has
been found to be expressed in 60% of ovarian cancers at the time of diagnosis (Slotman
and Rao, 1988). The work that I have described in this chapter focuses on novel
chemotherapeutics that selectively target cancer cells expressing the ER in the context
of ovarian cancer.
Our lab previously reported the synthesis of bifunctional compounds that displayed
selective toxicity in breast cancer cells that expressed the ER (Rink 1996). The design of
these compounds was based on the mechanistic understanding of cisplatin action.
These compounds consisted of a 2-phenylindole (2PI) group which serves as a ligand
for the ER, linked to N,N-bis(2-chloroethyl)aniline that can react with DNA to produce
covalent adducts. These drug-DNA adducts have the unique ability to form complexes
with the ER. We proposed that the formation of ER-DNA adduct complexes prevented
repair of the adducts in ER(+) cells and was responsible for the selective toxicity that we
observed in ER(+) cells. Since ER has been found to play a crucial role in ovarian
cancer, we hypothesized that such compounds may have similar effects in ovarian
cancer cell lines.
205
In this chapter the toxicity of two of our bifunctional compounds towards the ovarian
cancer cell lines Caov-3 and OVCAR-3 is investigated. While both compounds contain a
reactive N,N-bis(2-chloroethyl)aniline group, they have dissimilar 2-phenylindole groups
with high and low affinities for the ER. These compounds - 2PI-C6NC2-mustard (2PI;
high affinity for the ER) and 2PI(OH)-C6NC2-mustard (2PI(OH); low affinity for the ER) were tested in ovarian cancer cells, and by studying the responses to these compounds
we hoped to distinguish the role of the ER in mediating the biological effects of the
phenylindole compounds. Comparisons were also made between cellular response to
these compounds and cisplatin, the current front-line therapeutic in ovarian cancer. The
experiments in this chapter were a joint effort by Dr. Pei-Sze Ng and me, and references
will be made wherever relevant.
Materials and Methods
Chemicals:
The bifunctional compounds 2PI and 2PI(OH) used in these studies were synthesized in
the Essigmann laboratory by Pei-Sze Ng. Radiolabeled analogs of the 2PI and 2Pl(OH)
compounds were prepared by coupling [14C]-labeled nitrogen mustards (with a specific
activity of 26 mCi/mmol) to phenylindole carbonates. Details of the synthetic steps and
characterization of the compound have been described in a previous publication (Rink et
al., 1996). The synthesis has also been outlined in Figure 4.1. Cisplatin, 17p-estradiol
and propidium iodide (PI) were obtained from Sigma-Aldrich.
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Cell Culture:
Caov-3 and NIH:OVCAR-3 (referred to as OVCAR-3) cells were obtained from the
American Type Culture Collection (ATCC, Rockville, MD). Fetal Bovine Serum (FBS)
was obtained from Thermo Fisher Scientific (Logan, UT). OVCAR-3 cells were
maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 2.5 mg/mL glucose,
2 mM GlutaMax, 1 mM sodium pyruvate and 100 mM HEPES buffer, supplemented with
0.01 mg/mL bovine insulin (Invitrogen) and 10% FBS. Caov-3 cells were maintained in
DMEM (Invitrogen) supplemented with 10% FBS and 1 mM sodium pyruvate. All cell
lines were grown in a humidified 5% CO2/air atmosphere at 370C.
For growth inhibition assays, cells were seeded in 12-well plates at 5x10 4 cells per well.
Twenty four hours later, the test compounds dissolved in DMSO were added to the
medium. After 24 hours, the drug-containing medium was removed and the cells were
grown in fresh medium for an additional 24 hours. Cells were detached using
trypsin/EDTA solution (Invitrogen) and the number of cells in control and drug-treated
wells were determined using a Z1 Coulter Counter. The percent growth inhibition was
calculated as the ratio of cell number in treated to that in control wells multiplied by 100.
Significance values were calculated using unpaired t tests, with a two-tailed P value less
than 0.05 indicating significance. Growth inhibition assays were performed by Pei-Sze
Ng.
Relative Binding Affinity Assays:
Relative ligand binding affinities (RBAs) were determined by a competitive radioligand
binding assay using purified ER from a commercial source (ERa; Invitrogen).
[3H]-estradiol at 10 nM and ERa at 1.5 nM were combined with unlabeled competitors
207
and incubated for 12-18 hours at 40C. Receptor-ligand complexes were adsorbed to
hydroxyapatite and radioactivity was determined by scintillation counting. Binding
affinities were expressed as a percentage relative to estradiol (estradiol RBA =100%).
Binding assays were conducted by Pei-Sze Ng.
Immunoblot Analysis:
Whole cells extracts used for ER analyses were prepared by lysis of cells in SDS sample
buffer (62.5 mM Tris-HCI (pH 6.8), 2% SDS, 41.6 mM DTT, 10% glycerol, 0.01%
bromophenol blue). Whole cell extracts for cleaved caspase-3 were prepared by cell
lysis using RIPA buffer (Santacruz; 1X tris-buffered saline (TBS), 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM sodium orthovanadate, 1
mM NaF, 50 mM Tris-HCI (pH 7.4) and protease inhibitor cocktail (Sigma-Aldrich)).
Protein concentrations were determined using Bio-Rad RC-DC protein assay with
Bovine Serum Albumin (BSA) standard. Equal amounts of protein were loaded and
separated on SDS-polyacrylamide gels and transferred to Immobilon-P membranes
(Millipore, Bedford, MA). Membranes were blocked with 5% milk in TBS-Tween20
(TBS-T; 0.1% Tween20, 10 mM Tris (pH 7.4), 150 mM NaCl) and incubated with primary
antibodies overnight at 40C. The antibody against ER (mouse monoclonal) was obtained
from Upstate Biotechnology (Lake Placid, NY) and anti-cleaved caspase-3 antibody was
purchased from Cell Signaling Technologies (Beverly, MA). Anti-mouse and anti-rabbit
secondary antibodies conjugated to horseradish peroxidase were obtained from Cell
Signaling Technologies. Membranes were incubated with secondary antibodies for 1-2
hours, and antibody complexes formed with the secondary antibodies were visualized
using Enhanced Chemiluminescence reagents (SuperSignal West Femto, Pierce,
Rockford, IL).
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Cell-Cycle Analysis:
Cells were treated with test compounds for 30-32 hours. Both adherent and nonadherent cells were recovered, washed with cold phosphate-buffered saline (PBS) and
fixed in 100% ethanol overnight at 4*C. Ethanol-fixed cells were washed with 1% FBS in
PBS, resuspended at 106 cells/mL, and stained with 10 tg/mL Pl. Stained cells were
incubated with 0.5 pg/mL RNase A (DNase free; Roche Diagnostics) for 30 minutes at
370C. DNA content was determined using a Becton-Dickinson flow cytometer (BD
FACScan). Cell cycle data was analyzed using the MODFIT-LT 3.0 program to estimate
the percentage of cells in G1, S and G2/M phases of the cell cycle.
Single-Cell Alkaline Gel Electrophoresis (Comet assay):
Cells were treated with test compounds for indicated times. Alkaline comet assay was
performed as outlined in the Trevigen comet assay protocol using the reagents provided
in the Trevigen kit (Trevigen, Gaithersburg, MD).
Following treatment, cells were
trypsinized, resuspended in PBS at 105 cells/mL and irradiated on ice using a
GammaCell 40 Irradiator with a dose of 8 Gy. Irradiated cells were suspended in liquid
Low Melting Agarose (LMA) at 370C and plated onto a CometSlide (Trevigen). After the
agar solidified, cells were incubated in lysis solution (2.5 M NaCl, 100 mM EDTA, 10mM
Tris base (pH 10), 1%Sodium Lauryl Sarcosine, 0.01% Triton X-100) for an hour at 40C,
followed by incubation with 100 pL of 1 mg/mL Proteinase K (Roche Diagnostics) for an
hour at room temperature. Slides were then treated for 30 minutes with alkaline solution
(0.3 M NaOH, 1 mM EDTA, pH >13), followed by electrophoresis under alkaline
conditions (0.3 M NaOH, 1mM EDTA). Slides were then dried, stained with SYBR Green
dye, and photographed using a Nikon Eclipse epifluorescence microscope. Images
209
were analyzed using the Komet 4.2 Single Cell Gel Electrophoresis software (Kinetic
Imaging Limited, U.K.). A total of 100 cells per sample were analyzed, 50 per duplicate
slide. The Olive Tail Moment (OTM), defined as the product of the tail length and the
fraction of total DNA in the tail, was calculated for each cell by the software. OTM
incorporates a measure of both the smallest detectable size of migrating DNA (reflected
in the comet tail length) and the number of relaxed or broken pieces which is
represented by the intensity of DNA in the tail (Olive et al., 1990; Olive and Banath,
2006). Comet data were represented using boxplots plotted using SSC-Stat, a statistical
add-in for Excel.
Boxplot was used to give a graphical representation of the heterogeneity of the comet
data. The box contains the middle 50% of the data, the upper boundary of the box
indicates the
75 th
percentile of the data, and the lower boundary the
25 th
percentile. The
range of the middle two quartiles is called the interquartile range. The line in the box is
the median. The ends of the "whiskers" on the boxplot extend to a maximum of 1.5 times
the interquartile range. The average OTM values were indicated on the boxplot.
Percentage decrease in the average OTM was calculated for each treatment relative to
the untreated irradiated control, and significance values were calculated using the MannWhitney statistic (P < 0.001 was considered significant).
Adduct measurement using Accelerator Mass Spectrometry (AMS):
Cells were exposed to various doses of [14C]-labeled test compounds. At the end of the
incubation period, wells were washed once with fresh medium, followed by a wash with
PBS, and cells were trypsinized and collected. They were then lysed in 0.5 mL extraction
buffer containing HEPES (50 mM), NaCl (100 mM), EDTA (10 mM) and 0.8% SDS, and
210
incubated with RNase A (Roche Diagnostics; 0.9 mg/mL, 30 minutes at 370C) followed
by Proteinase K (Roche Diagnostics; 0.6 mg/mL, 2 hours at 370C). Samples were
subjected to phenol:chloroform extraction, and the aqueous phase was extracted once
more with chloroform. DNA was precipitated with 100% ethanol overnight at -200C, and
subsequently pelleted by centrifugation at 7000g for 15 min at 40C. The pellet was
washed with 70% ethanol, and dried in vacuo. DNA was dissolved in 0.2 mL water and
the concentration was estimated by absorbance at 260 nm. AMS analyses were
conducted at the Biological Engineering Accelerator Mass Spectrometry (BEAMS) Lab
at MIT by Paul Skipper and Rosa Liberman as described in Liberman, et al. (2004). DNA
solutions were applied directly to a CuO matrix used for sample combustion. A standard
consisting of a solution of [14C-methyl] bovine serum albumin was used to calibrate the
instrument. The amount of [14 C] in each sample was calculated from the peak area ratio
of the sample to the standard. All samples were analyzed at least twice. Readings were
obtained from AMS instrument as dpm/pg DNA and converted to adducts per million
bases. Two-tailed P values for AMS experiments were calculated using unpaired t tests,
and a value of P<0.05 was considered significant. The distribution of the [14 C]-labeled
drug in cells and medium was calculated using scintillation counting.
Results
2PI demonstrates almost a ten-fold higher binding affinity to the ER compared to
2PI(OH). The binding affinities of the 2-phenylindole compounds relative to that of 17pestradiol were measured. The structures of the two compounds and their RBA values
are shown in Figure 4.2. The 2PI compound shows excellent binding affinity for the ER,
211
binding at 57% the affinity of estradiol. The 2PI(OH) compound, which lacks the
5-hydroxy group on the ER recognition domain, only weakly interacted with the ER
(RBA=6). The binding affinities of the phenylindole compounds follow the same trend as
was observed with the published data on these compounds (Rink et al., 1996).
Caov-3 and OVCAR-3 express the ER. In order to test whether the putative interaction
between the 2-phenylindole moiety and ER could occur in treated cells, the ER
expression in the two cell lines was studied. On immunoblotting OVCAR-3 and Caov-3
cells for ER levels, the protein was detected in both the cell lines, as can be seen in
Figure 4.3. The breast cancer cell line MCF-7 was used as a positive control for ER
expression. The relative levels of the receptor were calculated using spot densitometry.
The levels of ER (relative to Caov-3 cells) were Caov-3: OVCAR-3: MCF-7 = 1: 0.8: 1.8.
MCF-7 cells expressed approximately twice the level of ER as the ovarian cancer cells.
This is in agreement with the levels reported in previous studies (Havrilesky et al., 2001).
2PI(OH) and 2PI exert equivalent toxicity in both Caov-3 and OVCAR-3 cells. The
cytotoxicity of the compounds 2P1 and 2PI(OH) was determined in comparison to
cisplatin in the two ovarian cancer cell lines. The results of the experiment are shown in
Figure 4.4. The cytotoxicity data demonstrated that Caov-3 cells were more sensitive to
all the compounds tested than OVCAR-3 cells. This observation is also consistent with
the reported data on the sensitivity of Caov-3 cells to cisplatin relative to OVCAR-3
(Serova et al., 2006).
In Caov-3 cells, all the three compounds seemed to induce similar levels of toxicity. The
cells showed a slightly enhanced sensitivity towards 2PI(OH) compared to 2PI and
cisplatin at the 5 pM dose, but all the other doses were equivalently cytotoxic. At doses
212
above 5 pM, the three compounds were able to achieve almost a 100% cell kill. In
OVCAR-3 cells, a similar trend was observed, although the cells were less sensitive to
the three compounds than Caov-3. At concentrations of 5 pM and 10 pM, 2PI(OH) was
more cytotoxic than both 2PI and cisplatin. Cisplatin was unable to achieve complete cell
killing even at the highest concentration tested (20 pM), in contrast to 2P and 2PI(OH).
Overall, in both the cell lines, the three compounds seemed to be equivalent in inducing
growth inhibition. The unexpected finding was that the 2PI(OH) compound was as toxic
as the 2PI compound in both the cell lines tested. The selective increased toxicity of 2PI
over 2PI(OH) that was previously seen in ER(+) breast cancer cells (Rink et al., 1996)
was not observed in either of the ovarian cancer cell lines tested.
2PI induces S-phase arrest in ovarian cancer cells. In order to study whether the
cytotoxicity of the phenylindole compounds was associated with changes in the cell
cycle patterns, the effect of our compounds on cell cycle progression of Caov-3 and
OVCAR-3 cells was evaluated by flow cytometry. The results are shown in Figure 4.5
(Caov-3) and Figure 4.6 (OVCAR-3). Both cell lines were treated with 2.5 pM of
compounds for 30-32 hours (one complete cell cycle). In the control cell population, a
higher S-phase fraction in Caov-3 cells (35%) than in OVCAR-3 cells (21 %) was
observed. A higher S-phase population in the untreated Caov-3 cells could increase its
susceptibility to DNA damage (Kolfschoten et al., 2000). This might explain the
observation that Caov-3 cells were more sensitive to treatment with our compounds and
cisplatin in comparison to OVCAR-3 cells.
In both the cell lines, we noticed consistent trends in the response to the three
compounds. 2PI and cisplatin treatments resulted in a marked increase of cells in the
213
S-phase compared to the control population. 2PI increased the S-phase population from
35% to 65% in Caov-3 cells and from 21% to 71% in OVCAR-3 cells. Cisplatin treatment
arrested 80% of Caov-3 cells and 46% of OVCAR-3 cells in the S-phase. This result is
consistent with the report that the cisplatin causes DNA damage and arrest cells in the
S-phase or the G2/M phase of the cell cycle (Nguyen et al., 1993; Sekiguchi et al., 1996).
In contrast, 2PI(OH) treatment caused only a slight increase in S-phase from 21%-39%
in OVCAR-3 cells, and no changes in cell cycle patterns were observed in Caov-3 cells
with drug treatment.
The cytotoxic profiles of the three drugs at this dose (2.5 pM) were similar to each other,
and were also equivalent in the two cell lines. The dissimilarity in the cell cycle
distributions with 2PI(OH) treatment compared to 2PI and cisplatin seems to indicate
that 2PI(OH) may be acting via a different mechanism from the other two compounds.
Comet assay detects crosslinks induced by 2PI in both the cell lines, and by
2PI(OH) in OVCAR-3 cells. Cells treated with the phenylindole compounds were
subjected to the modified alkaline comet assay (Olive and Banath, 1995; Olive and
Banath, 2006) to determine the presence of interstrand crosslinks (ICLs). Drug-treated
or mock-treated cells were .irradiated with 8 Gy dose to induce random DNA strand
breakage, followed by cell lysis and alkaline unwinding. The presence of ICLs retards
the migration of the irradiated DNA during alkaline electrophoresis, resulting in a
reduced Olive Tail Moment (OTM) compared to the mock-treated irradiated control. The
results of the experiment are shown in Figure 4.7 (Caov-3, 6 hours) and Figure 4.8
(OVCAR-3, 8 hours). The percent decrease in tail moments, which indicates the extent
of crosslink formation, is listed in Table 4.1.
214
In Caov-3 cells, treatment with cisplatin at 2.5 pM for 6 hours showed a significant
decrease in tail moment, and increasing the dose to 5 pM did not seem to change the
extent of crosslinking detected by the assay - both the doses of cisplatin induced
approximately 30% decrease in OTM. 2PI compound was also found to induce
crosslinking, although to a lesser extent than cisplatin. The 2.5 pM dose of 2PI induced a
20.8% decrease in OTM and the 5 pM dose a 13.1 % decrease in OTM compared to the
irradiated sample. The percent decrease in OTM induced by 2PI was significant at both
the doses, indicating that ICLs were being formed by the 2P1 compound.
In contrast, 2PI(OH) treatment did not induce any detectable crosslink formation in
Caov-3 cells. The interesting observation with 2PI(OH) treatment was that both the
doses induced a significant increase in OTM compared to the irradiated control. This
result can be explained by the possible formation of extensive single strand breaks or
induction of changes related to apoptosis such as DNA fragmentation with the treatment.
Both these situations would relax the DNA strands, leading to increased OTM in the
2PI(OH) treated samples compared to the untreated irradiated control. Therefore, the
effect we see is likely a net result of crosslinks and strand breaks, and an increase in
OTM indicates that the effect of the single strand breaks outweighs that of any crosslinks
that might have formed. The increase in OTM does not rule out ICL formation, it merely
eliminates the detection of the lesion with this assay.
All the three compounds formed crosslinks in OVCAR-3 cells (Figure 4.8, Table 4.1),
and the crosslinking was more potent in these cells than in Caov-3 cells. Cisplatin
displayed the highest extent of crosslinking - a significant decrease in OTM of 48% with
2.5 pM dose and 71% with 5 pM dose were observed. 2PI demonstrated a 54%
decrease in OTM with both the doses. 2PI(OH) also produced crosslinks in these cells,
215
although to a lesser extent than cisplatin and 2PI (a decrease in OTM of 25% at 2.5 pM
and 40% at 5 piM).
Crosslinking caused by the drugs could lead to a block in replication, triggering
checkpoint activation, and leading to cell cycle arrest. In this light, the results of the
comet assay seem to be consistent with the cell cycle experiments. In Caov-3 cells,
cisplatin and 2PI induced S-phase arrest and these drugs were also shown to form ICLs.
2PI(OH) does not show a change in the cell cycle distribution and no crosslinking ability
could be detected using the comet assay. A similar parallel was seen between the cell
cycle arrest patterns and crosslink formation in OVCAR-3 cells.
Similar adduct levels are detected with the test compounds using AMS in
OVCAR-3 and Caov-3 cells. We were interested in testing whether the phenylindole
compounds formed similar levels of DNA adducts in treated cells. AMS studies (results
shown in Figure 4.9) with radiolabeled 2PI and 2PI(OH) demonstrated that both the
compounds were capable of forming DNA adducts in treated cells. Doses from 0.5 IM to
5 pM were tested, and all the doses were observed to form similar number of DNA
adducts in both Caov-3 cells and OVCAR-3 cells. At the 5 IM dose there was an
abnormally low number of adducts in OVCAR-3 cells treated with 2PI(OH). It was
expected that the 5 pM dose would produce as many as, if not more adducts than the
2.5 pM dose of 2PI(OH). This anomalous response was not observed in the Caov-3 cells.
This data could not be explained, and we regarded this anomalous reading as an outlier
point. All the other doses from 0.5-3.5 pM in both cell lines seemed to demonstrate a
clear dose response to the drugs. Although some doses seemed to produce more
adducts with one compound than with the other, overall, we did not detect any significant
changes in adduct levels with the two compounds. The analysis of formation of DNA
216
adducts demonstrated that 2PI(OH) was as effective as 2PI in the formation of DNA
adducts. Both the cell lines also seemed to form an equivalent number of adducts with
the phenylindole compounds.
A stronger caspase-3 activation is observed with 2PI(OH) than with 2PI treatment.
Cells treated with 2PI and 2PI(OH) were immunoblotted for caspase-3 in order to
investigate whether apoptosis occurred as a result of treatment. The results are shown
in Figure 4.10. Caov-3 cells were treated with 5 pM and OVCAR-3 cells with 10 pM of
test compounds for 15 hours. In both the cell lines, it was observed that treatment with
cisplatin and 2PI(OH) induced a strong activation of procaspase-3 (32 kDa) to form the
cleaved 17 kDa caspase-3 product, which was specifically detected by the antibody. The
2PI compound was also able to induce the activation of cleaved caspase-3, but only
weakly. The difference in the activation of caspase-3 between 2PI and 2PI(OH) is
consistent with the cytotoxicity studies; in Caov-3 cells at the 5 pM dose of the
compounds, and in OVCAR-3 cells at the 10 pM dose, 2PI(OH) was significantly more
cytotoxic than 2Pl. This difference in cytotoxicity is likely reflected in the caspase-3
activation - 2PI(OH) induces a much stronger apoptotic signal (as detected by the
cleavage of procaspase-3 to the 17 kDa fragment) than the 2PI compound.
Discussion
Using the lessons learned from cisplatin, the Essigmann lab sought to improve the
therapeutic index of antitumor agents by targeting cancers that aberrantly expressed the
ER. The principle behind the drug design was that by combining a DNA damaging
functionality and a protein recognition moiety specific for a cancer cell in the same
217
molecule, selective toxicity towards cancer cells could be achieved. This chapter deals
with the study of the first set of rationally designed drugs developed in the Essigmann
laboratory. These agents (2-phenylindole compounds, Figure 4.2) target cancers that
express the ER. Rink et al. (1996) have described in detail the synthesis and
characterization of the phenylindole compounds.
The compound 2PI-C6NC2-mustard (2PI) contains the 2-phenylindole moiety that
serves as a ligand for the ER (RBA of 57 relative to estradiol), and a DNA alkylating
aniline mustard moiety that is capable of reacting covalently with DNA. 2PI(OH)-C6NC2mustard was synthesized as a control compound that has poor affinity for the ER (RBA =
6, Figure 4.2) as a result of only a monohydroxylation of the phenylindole moiety (von
Angerer et al., 1984). Our hypothesis of the mechanism of action of 2PI is that it forms
DNA lesions that attract the transcription factor ER. As explained in Rink et al. (1996),
association with the ER could either shield the DNA adduct from repair or titrate away
the transcription factor from its normal site of binding, thereby inducing toxicity. The
2PI(OH) compound is designed to not interact with the ER, and we expected it to be less
toxic than the 2PI compound for this reason. We had seen promising results consistent
with the above mechanism with the phenylindole compounds in breast cancer cells; it
was observed that the 2PI compound was more toxic to MCF-7 breast cancer cells than
its analog that does not interact with the ER. This result supported our hypothesis that
the interaction with ER is crucial to the toxicity of our compound. The favorable toxicity
profiles of the phenylindole compounds in breast cancer cells prompted us to investigate
whether a similar response towards the compounds could be detected in ovarian cancer
cells.
218
Cytotoxicity assays revealed that overall, Caov-3 cells were more sensitive to cisplatin,
2PI and 2PI(OH) than OVCAR-3 cells. The unexpected finding from the growth inhibition
studies was that the 2PI(OH) control compound was as toxic to the cells as the 2PI
compound. We expected that the ovarian cells, similar to the breast cancer cells tested
earlier (Rink et al., 1996), would be more sensitive to the 2PI compound than to 2PI(OH).
The differences in the response of the ovarian cells indicate that the two compounds are
likely acting via different mechanisms in these cells.
Although similar toxicity towards the two compounds was observed in the ovarian cancer
cells, the apoptotic marker caspase-3 was found to be much more strongly induced by
2Pl(OH) compared to 2PI, indicating that the 2PI(OH) compound clearly induces
apoptosis. This finding shown that 2PI(OH) and 2PI are clearly different in the way they
induce cell death. Alternative modes of cell death induced by 2PI (in addition to
apoptosis) might be able account for the toxicity of the compound. In this regard, we can
draw a parallel between E2-7a-C6NC2-mustard (which contains estradiol as the ER
binding domain) and the 2PI compound. We had discussed the cytotoxicity and cell
death patterns induced by E2-7a in ovarian cancer cells in Chapter 2, and it was found
that the compound induces cell death predominantly through the pathway of autophagy,
and that no caspase-3 activation could be detected in ovarian cells treated with E2-7a.
Since 2PI and E2-7ax are similar in their binding ability to DNA and both the compounds
alkylate DNA, it is reasonable to expect similar effects in their manner of inducing cell
death. Since 2PI does not seem to induce apoptosis, it is possible that the compound
induces another form of cell death - autophagy - which could account for the toxicity of
the compound. This possibility could also account for the absence of a strong cleaved
caspase-3 signal with 2PI treatment. Investigations along these lines would help piece
together the caspase-3 activation and the toxicity data of the phenylindole compounds;
219
these studies would also help understand the different mechanisms of action of the
phenylindole compounds.
The cytotoxicity of 2PI(OH) in the cisplatin-resistant ovarian cancer cells was intriguing
and its mechanism of action was investigated further by studying the progression of cell
cycle. In our studies, 2PI, similar to cisplatin, caused a potent S-phase arrest in Caov-3
and OVCAR-3 cells. 2PI(OH), however, did not cause any noticeable changes in the cell
cycle patterns in Caov-3 cells and caused a slight S-phase arrest in OVCAR-3 cells.
These observations can be explained by one of the two possibilities: (i) 2PI(OH)
resulted in little or no DNA damage in comparison to cisplatin and 2PI, or (ii) the damage
induced by 2PI(OH) went undetected or was not given sufficient time to be repaired,
hence it bypassed the cell cycle checkpoints to directly induce toxicity. In either case, it
seems likely that 2PI and 2PI(OH) target the cells through different mechanisms, which
may or may not involve the ER.
Both the phenylindole compounds contain a nitrogen mustard moiety capable of forming
monoadducts and crosslinks, and the repair of such kinds of DNA damage could lead to
delay in cell cycle progression (Brookes and Lawley, 1961; Meyn and Murray, 1986). We
considered the possibility that the two compounds might be inducing different levels of
DNA damage in treated cells, leading to the difference in cell cycle responses. In order
to test this hypothesis, AMS and comet assay were employed to detect DNA adducts
and ICLs respectively.
The AMS experiments enabled the detection of drug-DNA adducts in populations treated
with [14C]-2-phenylindole compounds. It was found that the two compounds formed a
similar number of DNA adducts in both the cell lines (Figure 4.9). Moreover, the dose
220
response curve showed that the level of adducts was equivalent in Caov-3 and
OVCAR-3 cells. Overall, the results of the AMS experiment indicated that the difference
in the mechanism of action of 2PI and 2PI(OH) is not likely to stem from the differences
in their abilities to form DNA adducts.
The ability of the phenylindole compounds to form ICLs in treated Caov-3 and OVCAR-3
cells was examined using the comet assay. The comet assay results in Caov-3 cells
(Figure 4.7) clearly demonstrated the ability of 2PI to form ICLs in these cells. ICL
formation caused by the control compound could not be detected in Caov-3 cells. In fact,
our observations indicate that the presence of any ICLs in these cells following 2PI(OH)
treatment might have been masked by cell changes related to apoptosis (for example,
DNA fragmentation) or by the formation extensive single strand breaks. We observed a
similar trend in OVCAR-3 cells - both the compounds were found to induce ICL
formation in these cells, although to different extents. The results of the comet assay
indicate that the compounds differed in their ability to form crosslinks in the cells, or in
their ability to form single strand breaks, or a combination of both. It was not evident
whether the ER played a role in the toxicity of these compounds in the ovarian cancer
system.
The 2PI compound displayed DNA damage characteristics that are distinctive of an
aniline mustard (Sunters et al., 1992) - it caused cytotoxicity and arrested both the cell
lines in the S-phase of the cell cycle. The arrest pattern can be explained by the
formation of ICLs leading to a block in replication, directly causing S-phase arrest in the
cells. The presence of DNA damage, crosslinks and S-phase arrest could explain the
observed toxicity of the compound.
221
The control compound 2PI(OH) did not seem to function as per its intended mechanism
of action. Guided by the observation that the compound damaged DNA and induced
apoptosis in treated cells but did not cause any changes in the cell cycle distribution, we
considered certain alternative routes by which 2PI(OH) may be inducing toxicity. One
possible explanation stems from the hypothesis that the compound interferes with cell
cycle checkpoint machinery to suppress the activation of checkpoint proteins that are
required to halt the cell cycle in response to DNA damage. The checkpoint abrogation by
2PI(OH) could underlie the difference in modes of action of the two phenylindole
compounds. It is possible that the following 2PI(OH) treatment, cells proceed through the
cell cycle despite their DNA damage, and therefore, are likely to be very sensitive to the
compound.
There are reports in the literature that cells that have not been arrested in S or G2
phases after exposure to a damaging agent and still enter mitosis with damaged DNA
undergo cell death rapidly (Shapiro and Harper, 1999). Drugs that facilitate this kind of
checkpoint abrogation could be extremely cytotoxic. Agents that inhibit cell cycle arrest
elicited by DNA damage, and thereby sensitize cells to cisplatin and other alkylating
drugs have been reported in the literature (Lau and Pardee, 1982; Schlegel and Pardee,
1986; Sugiyama et al., 2000). Furthermore, it has been shown that the sensitization of
cells is especially selective for p53-deficient cells (Waldman et al., 1996). Cells that have
compromised p53 are able to enter mitosis following DNA damaging treatments, and
mitotic entry before the damage repair leads to cell death. The p53 tumor suppressor
protein is required for G1 arrest induced by DNA damage in most cells, but usually not
for arrest in S or G2. Normal cells (p53 positive) arrest mainly in G1, whereas p53negative tumor cells arrest in S or G2 in response to DNA damage. Therefore, the
222
abrogation of these checkpoints would make the p53 deficient cells extremely sensitive
to DNA damage.
One of the first compounds that was shown to abrogate S and G2 checkpoints and
induce selective killing in p53-deficient cells is 7-hydroxystaurosporine (UCN-01), a
protein kinase inhibitor (Bunch and Eastman, 1996; Bunch and Eastman, 1997; Wang et
al., 1996). UCN-01 abrogates S and G2 checkpoints reportedly due to its inhibition of
both Chk1 activity and the Cdc25C pathway (Graves et al., 2000). It has been shown
that UCN-01 enhances the toxicity of cisplatin by abrogating the cell cycle arrest caused
by cisplatin (Bunch and Eastman, 1996; Bunch and Eastman, 1997). This report
suggests a possible mechanism of 2PI(OH) action - the compound might be capable of
abrogating the checkpoint response to the arrest caused by its own DNA alkylating
moiety. This would lead to enhanced sensitivity to the compound especially in p53
mutant cells. In our case, both Caov-3 and OVCAR-3 cells are p53 mutant (Hagopian et
al., 1999; Wolf et al., 1999), and have been shown to be vulnerable to the effects of
2PI(OH). In effect, our compound might be assuming the roles of both UCN-01 and
cisplatin in this system. It could be interacting with the cell cycle checkpoint machinery to
inhibit cell cycle arrest, and thereby enhancing the toxicity caused by the aniline mustard
moiety.
Understanding the basis for checkpoint abrogation by the 2PI(OH) compound requires
further investigations. These studies will doubtless help elucidate the basis of the
differences in the mechanisms of action of 2PI and 2PI(OH). We postulate that the
underlying mechanism of 2PI(OH) action might involve the interaction with cell cycle
regulatory proteins, leading to abrogation of cell cycle checkpoints, whereas the 2PI
compound induces DNA damage, cell cycle arrest and cytotoxicity. The role of ER in the
223
toxicity of either of the compounds needs to be investigated further as well. It is possible
that the ER does not play a major role in the inhibition of cell proliferation with one or
more of the phenylindole compounds in these cell lines, in contrast to the ER(+) breast
cancer cell line MCF-7. Since the only difference between the 2PI(OH) and 2PI is the
absence of the 5-hydroxy group in the phenylindole moiety in 2PI(OH), identification of
the biological interactions of this portion of the molecule with the cellular machinery
could shed light on these mechanisms.
Conclusions
This chapter describes some interesting observations that were made with the
cytotoxicity and DNA damage studies of the 2-phenylindole compounds 2PI and 2PI(OH).
The DNA damage profiles and the patterns of cell cycle arrest observed with the two
compounds underscore the differences in the modes of action of the two compounds in
the p53 mutant ovarian cell lines tested. In this light, 2PI might be inducing toxicity as per
its intended mechanism, by interacting with the ER and forming adducts that are
shielded from repair, thus inducing toxicity to treated cells. The role of ER in the toxicity
of 2P1 needs to be delved into in further detail, and this will be one of the main aims of
future investigations with this compound. 2PI(OH) displays the potential to combine the
effects of a checkpoint abrogator and a DNA alkylating agent in one molecule, and the
interaction of the compound with checkpoint machinery needs to be investigated. Both
the compounds demonstrate promise as potent anti-tumor agents, and the selectivity of
these compounds in different cellular contexts (ER (+)/(-) or p53 (+)/(-)), if established,
could help strengthen the basis of their therapeutic efficacy.
224
Detection of crosslinks using comet assay:
Percentage decrease in OTM in Caov-3 and
OVCAR-3 cells
Percent decrease in OTM
Treatment
Caov-3
OVCAR-3
Cisplatin 2.5 pM
32 (*)
48(*)
Cisplatin 5.0 pM
2PI 2.5 pM
35 (*)
71(*)
21 (*)
54(*)
2PI 5.0 pM
13(*)
54(*)
2PI(OH) 2.5 pM
-
25(*)
2PI(OH) 5.0 pM
-
40(*)
(*) - significant decrease in OTM
(P<0.001)
Table 4.1: Percent decrease in OTM vs. treatment. The
corresponding OTM values are plotted in Figure 4.7
(Caov-3) and Figure 4.8 (OVCAR-3). The percentage
decrease in OTM is relative to the OTM of untreated
irradiated sample. Significance is calculated using MannWhitney test. The percent decrease in OTM indicates
extent of crosslink formation.
225
Synthetic scheme of 2-Phenylindole compounds:
2PI (14a) and 2PI(OH) (14b) - Part 1
R
0
/ \O
-i
i -
OH
H 2N
H
4
4a R1 =R2= OH
iy
4bR1=H, R2=OH
V
+
H
R2
NN
H
3
la-3a R R=R
2 =OCH3
lb-3bRi = H, R 2 = OCH 3
vi, vii
R3
R\i
R1
iii
NN
Br
R1
R2
R1
NN
R2
H
5
5a-6a Rj= R2= OCH2OCH 3
5b-6b Ri= H, R2 = OCH 2OCH 3
NH
NN
\2-
R1A
R1
C 6H12
b6H12
N-R
C6 H12
I
I
N-R
3
N-R3
3
t 8
HO
R2
9
0
10
8a-10a R1= R2= OCH 2OCH 3, R3= PO(C6 ,H5 ) 2
8b-10b R1= H, R2 =OCH 2OCH 3, R3 = PO(C 6 H 5)2
Conjugation of nitrogen mustard to ligand
0
H2N
HO
xi i
+
10
2
N
NI
R
N
R4
Ila, lb R4=Cl
R f4 12b
NR
4
"1
13
13a Ri= R2 = OCH 2OCH 3, R3 = PO(C4H5 )2 , R4= Cl
13b R1 = H, R2= OCH 2OCH 3, R3 = PO(CeH5) 2 , R4 = Cl
12a, 12bR4 = Cl
1xiii
R
R2
NN
R4
H
SNCH12N
14a Rr' R2 = OH, R4
14b R1= H, R2 = OH,
= C1
R4 =
O
ON
Cl
/H
M
14
Figure 4.1A: Synthetic scheme of 2PI-C6NC2-mustard and
2PI(OH)-C6NC2-mustard compounds. Intermediates are
labeled in Figure 4.1B
226
Synthetic scheme of 2-Phenylindole compounds:
2PI (14a) and 2PI(OH) (14b) - Part 2
(i) Compound 1 (1 eqv.), CuBr2 (2.4 eqv.), EtOAc, 900C (ii)
Compound 2 (1 eqv.), p-anisidine (3.3 eqv.), N, N
dimethylaniline, 2000C (iii) Compound 3 (1 eqv.), BBr 3 (3.3
eqv.), CH2Cl2 , -780C to RT overnight (iv) Compound 4 (1 eqv.),
NaH (2.5 eqv.), monomethylbromoether (2.5 eqv.), THF, -780C
to RT (v) Compound 5 (1 eqv.), 1,6-dibromohexane (6 eqv.),
NaH (2 eqv.), DMF, -780C to RT overnight. (vi) Aminoethanol
(2 eqv.), diphenylphosphinic chloride (1 eqv.),
diisopropylethylamine (1.3 eqv.),CH 2Cl2, OC to RT. (vii) Tertbutyldimethylsilyl chloride (1.2 eqv.), DMF, imidazole, RT (viii)
Compound 6 (1 eqv.), 7 (1.7 eqv.), NaH (2.8 eqv.),
tetrabutylammonium bromide (0.16 eqv.), benzene, reflux 2 hr
(ix) Compound 8 (1 eqv.), tertbutylammonium fluoride (2 eqv.),
RT. (x) Compound 9 (1 eqv.), p-nitrophenylchloroformate (2
eqv.), diisopropylethylamine (2 eqv.), CH2Cl2 , RT, 3 hr.(xi)
Compound 11 (1 eqv.), ethyl chloroformate (1.2 eqv.),
triethylamine (1.2 eqv.), sodium azide (2 eqv.) 0CC, acetone,
reflux in toluene, 8N HCI, 10 min. (xii) Compound 12 (2 eqv.),
compound 10 (1 eqv.), diisopropylamine (4 eqv.),
tetrahydrofuran, reflux, 3 h (xiii) Compound 13, HCI, methanol.
Figure 4.1 B: Intermediates in the synthetic scheme of
2PI-C6NC2-mustard and 2PI(OH)-C6NC2-mustard
compounds.
227
Structures and relative binding affinities (RBAs)
of phenylindole compounds
Linker
I
R
H
OYN
N
N-"O
H
N
H
OH
I
I~C
Ligand for
estrogen receptor
Compounds
R
Alkylating
group
RBA (relative
Putative cellular
to estradiol)
interaction
(%)
2PI
OH
57
DNA, ER
2PI(OH)
H
6
DNA
Figure 4.2:Structures and relative binding affinities of the
2-Phenylindole compounds. The two compounds differ
only intheir 5-hydroxy group in the phenylindole domain.
Binding assays were conducted using purified ER obtained
from Invitrogen. RBAs were calculated on a percentage
scale relative to 17p-estradiol.
228
ER status of relevant cell lines
0
ERa (66 kDa) e
0
C
C
MCF-7
W mow& W
IMO
Actin amme ="""
o - OVCAR-3
C - Caov-3
Figure 4.3: ER status of OVCAR-3,Caov-3 cells and
MCF-7 (positive control). Actin was used as a loading
control.OVCAR-3 and Caov-3 cells express the ER, as
does the positive control MCF-7. The relative ratio of ER in
the three cell lines was determined by spot densitometry to
be Caov-3 : OVCAR-3: MCF-7 = 1 : 0.8: 1.8
229
2PI and 2PI(OH) cause growth inhibition of
Caov-3 and OVCAR-3 cells
A
Caov-3 cells
100
80
0
* 2PI(OH)
-- PCpI
--a- Cisplatin
60
CD,
C
40
20
0
10
5
15
20
Dose inuM
OVCAR-3 cells
100
0
+ 2PI(OH)
-A- 2P
-U- Cisplatin
80
L-
0)
60
(D,
40
20
0
10
5
15
20
Dose inuM
Figure 4.4: Growth inhibition curves for Caov-3 and
OVCAR-3 cells. Cells were treated with test compounds for
24 hours, followed by 24 hour incubation infresh medium.
Percent growth iscalculated relative to untreated control.
Values represent average of three independent
experiments, and error bars are standard deviations.
230
Cell cycle distribution in Caov-3 cells treated
with test compounds
Untreated
Cisplatin 2.5 piM
0.0
6.5
19.6
34.8
58.7
80.5
G1
G2/M
2PI 2.5 IM
2PI(OH) 2.5 piM
0.0
3.7
35.5
42.2
64.5
54.1
Figure 4.5: Pie-charts representing the cell cycle patterns
in Caov-3 cells treated with 2.5 pM dose of cisplatin, 2PI
and 2PI(OH) for 30 hours. Numbers around the pie-charts
represent percentage of cells in the corresponding phase
of cell cycle. Cisplatin and 2PI induce a potent S-phase
arrest, whereas 2PI(OH) does not seem to change the cell
cycle distribution.
231
Cell cycle distribution in OVCAR-3 cells treated
with test compounds
Cisplatin 2.5 p.M
Untreated
0.0
6.0
21.5
45.9
54.2
G1
72.6
S
G2/M
2PI 2.5 p.M
0.1
2PI(OH) 2.5 pM
8.2
29.
38.8
53.0
70.7
Figure 4.6: Pie-charts representing the cell cycle patterns
in OVCAR-3 cells treated with 2.5 pM dose of cisplatin, 2PI
and 2PI(OH) for 30 hours. Numbers around the pie-charts
represent percentage of cells inthe corresponding phase
of cell cycle. All the three compounds induce arrest inthe
S-phase, although to different extents.
232
-Chapter 4-
Detection of interstrand crosslink formation in
Caov-3 cells using comet assay
OTM vs. treatment: Caov-3 cells
20-
16 -9
E14 -
# Treatment
0~
12
7.7
.
=710-
1 Untreated
2 Irradiated 8Gy
3 Cis 2.5 pM+Irr
4 Cis 5.0 pM+Irr
5 2PI 2.5pM+Irr
T
--
O-
0
7.3
10
8
2
12.
*
11.3
18
62PI 5.0 pM+Irr
7 2PI(OH)2.5pM+Irr
1
8 2PI(OH)5pM+1rr
-
0.8
2
3
4
5
6
7
8
Figure 4.7: Boxplots showing distribution of tail moments
in Caov-3 cells. The numbers above the boxes represent
average OTM value. Both cisplatin and 2PI treatments
(starred entries) result in a significant decrease in OTM
compared to the irradiated control sample (P<0.001).
2PI(OH) did not induce a decrease in OTM, but increased
the OTM significantly (P<0.001).The percent decrease in
OTM is tabulated in Table 4.1.
233
-Chapter 4-
Detection of interstrand crosslink formation in
OVCAR-3 cells using comet assay
OTM vs. treatment: OVCAR-3 cells
20
18
10.7
16
# Treatment
*
8.0
*14
E
6.4
*
o 12
5.0 5.0
=10
*
3.1
.
-
_
6
1 Irradiated 8Gy
2 Cis 2.5 iM+Irr
3 Cis 5.0 pM+Irr
4 2PI 2.5 pM+lrr
5 2PI5.0 gM+Irr
6 2PI(OH)2.5pM+Irr
7 2PI(OH)5pM+Irr
42-
0
1
2
3
4
5
6
7
Figure 4.8: Boxplots showing distribution of tail moments
in OVCAR-3 cells.The numbers above the boxes represent
average OTM value. Cisplatin, 2PI and 2PI(OH) (starred
entries) treatments result ina significant induction of
crosslinks, although to different extents (P<0.001) . The
percent decrease in OTM istabulated inTable 4.1.
234
Drug-DNA adducts in Caov-3 and OVCAR-3 cells
detected by AMS
Caov-3 cells
OVCAR-3 cells
25
25
C,)
C,)
a)
C
0
20
20
4 2PIOVCAR-3
-*-2PI(OH)OVCAR-3
15
E
a,)
10
10
5
5
0
0.0 1.0 2.0 3.0 4.0 5.0
0.0 1.0 2.0 3.0 4.0 5.0
Dose in uM
Dose in uM
Treatment
dose in
piM
Adducts per million bases
Caov-3
2PI
Caov-3
2PI(OH)
OVCAR-3
2PI
OVCAR-3
2PI(OH)
1.52
0.55
1.29
1.71
7.43
0.5
1.0
2.5
1.15
1.15
1.66
2.58
6.96
3.5
5.0
6.96
14.7
7.43
23.6
1.48
4.01
11.3
17.4
16.1
7.79
Figure 4.9: Adducts per million bases following treatment
with various radiolabeled drugs measured using AMS.
Error bars represent standard deviation. The average
number of adducts per million bases vs. treatment has
been represented in the table above. Both the drugs form
similar number of adducts in the two ovarian cell lines.
235
Caspase-3 activation by cisplatin and
phenylindole compounds
Cis 2PI(OH) 2PI
Caov-3
Cleaved
caspase-3
OVCAR-3
Figure 4.10: Western blot for anti-cleaved caspase-3
antibody in cell extracts treated with cisplatin and the
compounds 2PI and 2PI(OH). Caov-3 cells were treated
with 5 pM and OVCAR-3 cells with 10 pM of test
compounds for 15 hours. Equal amounts of protein
(25pg/lane) were loaded ineach lane. Cisplatin and
2PI(OH) induce a strong activation of caspase-3 whereas
the 2Pl compound only mildly induces the formation of
cleaved caspase-3. Cleavage of caspase-3 can be
correlated to the extent of apoptosis inthese cells.
236
Reference List
1.
Abend M (2003) Reasons to reconsider the significance of apoptosis for cancer
therapy. Int J Radiat Biol 79:927-941
2.
Agarwal R, Kaye SB (2003) Ovarian cancer: strategies for overcoming resistance
to chemotherapy. Nat Rev Cancer 3:502-516
3.
Ahn HJ, Kim YS, Kim JU, Han SM, Shin JW, Yang HO (2004) Mechanism of taxolinduced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem
91:1043-1052
4.
Ali S, Metzger D, Bornert JM, Chambon P (1993) Modulation of transcriptional
activation by ligand-dependent phosphorylation of the human oestrogen receptor
A/B region. EMBO J 12:1153-1160
5.
American Cancer Society (2008) Cancer Facts and Figures.
6.
Arai M, Utsunomiya J, Miki Y (2004) Familial breast and ovarian cancers. Int J Clin
Oncol 9:270-282
7.
Araujo SJ, Nigg EA, Wood RD (2001) Strong functional interactions of TFIIH with
XPC and XPG in human DNA nucleotide excision repair, without a preassembled
repairosome. Mol Cell Biol 21:2281-2291
8.
Argento M, Hoffman P, Gauchez AS (2008) Ovarian cancer detection and
treatment: current situation and future prospects. Anticancer Res 28:3135-3138
237
9.
Armitage P, Doll (1954) The age distribution of cancer and a multi-stage theory of
carcinogenesis. Br J Cancer 8:1-12
10. Asahina H, Kuraoka 1,Shirakawa M, Morita EH, Miura N, Miyamoto I, Ohtsuka E,
Okada Y, Tanaka K (1994) The XPA protein is a zinc metalloprotein with an ability
to recognize various kinds of DNA damage. Mutat Res 315:229-237
11.
Balcome S, Park S, Quirk Dorr DR, Hafner L, Phillips L,Tretyakova N (2004)
Adenine-containing DNA-DNA cross-links of antitumor nitrogen mustards. Chem
Res Toxicol 17:950-962
12. Bandera CA, Ye B, Mok SC (2003) New technologies for the identification of
markers for early detection of ovarian cancer. Curr Opin Obstet Gynecol 15:51-55
13. Bardin A, Hoffmann P, Boulle N, Katsaros D,Vignon F, Pujol P, Lazennec G (2004)
Involvement of estrogen receptor beta in ovarian carcinogenesis. Cancer Res
64:5861-5869
14.
Barth H, Hoffmann 1,Klein S, Kaszkin M, Richards J, Kinzel V (1996) Role of
cdc25-C phosphatase in the immediate G2 delay induced by the exogenous
factors epidermal growth factor and phorbolester. J Cell Physiol 168:589-599
15.
Bast RC, Jr., Klug TL, St JE, Jenison E, Niloff JM, Lazarus H,Berkowitz RS,
Leavitt T, Griffiths CT, Parker L, Zurawski VR, Jr., Knapp RC (1983) A
radioimmunoassay using a monoclonal antibody to monitor the course of epithelial
ovarian cancer. N Engl J Med 309:883-887
16.
Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate
ribonuclease in the initiation step of RNA interference. Nature 409:363-366
238
17.
Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a
specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3-14
18.
Bishop JM (1991) Molecular themes in oncogenesis. Cell 64:235-248
19.
Biswas DK, Singh S, Shi Q, Pardee AB, Iglehart JD (2005) Crossroads of estrogen
receptor and NF-kappaB signaling. Sci STKE 2005:e27
20.
Bonadonna G, Brusamolino E, Valagussa P, Rossi A, Brugnatelli L, Brambilla C,
De LM, Tancini G, Bajetta E, Musumeci R, Veronesi U (1976) Combination
chemotherapy as an adjuvant treatment in operable breast cancer. N Engl J Med
294:405-410
21.
Bookman MA, Greer BE, Ozols RF (2003) Optimal therapy of advanced ovarian
cancer: carboplatin and paclitaxel versus cisplatin and paclitaxel (GOG158) and an
update on GOG0182-ICON5. Int J Gynecol Cancer 13 Suppl 2:149-155
22.
Borini A, Rebellato E (2008) Focus on breast and ovarian cancer. Placenta 29
Suppl B:184-190
23.
Botti J, Djavaheri-Mergny M, Pilatte Y, Codogno P (2006) Autophagy signaling and
the cogwheels of cancer. Autophagy 2:67-73
24.
Boulikas T, Vougiouka M (2004) Recent clinical trials using cisplatin, carboplatin
and their combination chemotherapy drugs (review). Oncol Rep 11:559-595
25.
Bowler J, Lilley TJ, Pittam JD, Wakeling AE (1989) Novel steroidal pure
antiestrogens. Steroids 54:71-99
239
26. Brandenberger AW, Lebovic DI, Tee MK, Ryan IP,Tseng JF, Jaffe RB, Taylor RN
(1999) Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial
and endometriosis-derived stromal cells. Mol Hum Reprod 5:651-655
27.
Brandenberger AW, Tee MK, Jaffe RB (1998) Estrogen receptor alpha (ER-alpha)
and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma
and ovarian cancer cell lines: down-regulation of ER-beta in neoplastic tissues. J
Clin Endocrinol Metab 83:1025-1028
28. Brookes, Lawley PD (1961) The reaction of mono- and di-functional alkylating
agents with nucleic acids. Biochem J 80:496-503
29.
Brown JM, Attardi LD (2005) The role of apoptosis in cancer development and
treatment response. Nat Rev Cancer 5:231-237
30.
Brown SJ, Kellett PJ, Lippard SJ (1993) lxrl, a yeast protein that binds to
platinated DNA and confers sensitivity to cisplatin. Science 261:603-605
31.
Bruhn, Toney JH, Lippard SJ (1990) Biological processing of DNA modified by
platinum compounds., 38 edn
32. Bucourt R,Vignau M, Torelli V (1978) New biospecific adsorbents for the
purification of estradiol receptor. J Biol Chem 253:8221-8228
33. Bunch RT, Eastman A (1996) Enhancement of cisplatin-induced cytotoxicity by 7hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res
2:791-797
240
34.
Bunch RT, Eastman A (1997) 7-Hydroxystaurosporine (UCN-01) causes
redistribution of proliferating cell nuclear antigen and abrogates cisplatin-induced
S-phase arrest in Chinese hamster ovary cells. Cell Growth Differ 8:779-788
35.
Burger RA (2007) Experience with bevacizumab in the management of epithelial
ovarian cancer. J Clin Oncol 25:2902-2908
36.
Chabner BA, Longo DL (2009) Cancer Chemotherapy and Biotherapy
37.
Chabner BA, Roberts TG, Jr. (2005) Timeline: Chemotherapy and the war on
cancer. Nat Rev Cancer 5:65-72
38.
Chatterjee M, Mohapatra S, lonan A, Bawa G, li-Fehmi R, Wang X, Nowak J, Ye B,
Nahhas FA, Lu K, Witkin SS, Fishman D, Munkarah A, Morris R, Levin NK, Shirley
NN, Tromp G, Abrams J, Draghici S, Tainsky MA (2006) Diagnostic markers of
ovarian cancer by high-throughput antigen cloning and detection on arrays. Cancer
Res 66:1181-1190
39.
Chen Z, Fadiel A, Jia JF, Sakamoto H, Carbone R, Naftolin F (1999) Induction of
apoptosis and suppression of clonogenicity of ovarian carcinoma cells with
estrogen mustard. Cancer 85:2616-2622
40.
Clark (2006) Molecular targeted drugs and growth factor receptor inhibitors. In:
Chabner BA, Longo DL (eds)
41.
Clarke PG (2002) Apoptosis: from morphological types of cell death to interacting
pathways. Trends Pharmacol Sci 23:308-309
241
42.
Clinton GM, Hua W (1997) Estrogen action in human ovarian cancer. Crit Rev
Oncol Hematol 25:1-9
43. Coezy E, Borgna JL, Rochefort H (1982) Tamoxifen and metabolites in MCF7 cells:
correlation between binding to estrogen receptor and inhibition of cell growth.
Cancer Res 42:317-323
44.
Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS (1997) Tissue
distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and
estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and
ERalpha-knockout mouse. Endocrinology 138:4613-4621
45. Cowley SM, Hoare S, Mosselman S, Parker MG (1997) Estrogen receptors alpha
and beta form heterodimers on DNA. J Biol Chem 272:19858-19862
46. Cunat S, Hoffmann P, Pujol P (2004) Estrogens and epithelial ovarian cancer.
Gynecol Oncol 94:25-32
47. Cunat S, Rabenoelina F, Daures JP, Katsaros D, Sasano H, Miller WR,
Maudelonde T, Pujol P (2005) Aromatase expression in ovarian epithelial cancers.
J Steroid Biochem Mol Biol 93:15-24
48.
DaSilva JN, van Lier JE (1990) Synthesis and structure-affinity of a series of 7
alpha-undecylestradiol derivatives: a potential vector for therapy and imaging of
estrogen-receptor-positive cancers. J Med Chem 33:430-434
49. De BK, Vanden BW, Haegeman G (2006) Cross-talk between nuclear receptors
and nuclear factor kappaB. Oncogene 25:6868-6886
242
50.
Deng CX (2006) BRCA1: cell cycle checkpoint, genetic instability, DNA damage
response and cancer evolution. Nucleic Acids Res 34:1416-1426
51.
DeVita VT, Hellman S, Rosenberg SA (1997) Princples and Practice of Oncology,
5 edn
52.
Donahue BA, Augot M, Bellon SF, Treiber DK, Toney JH, Lippard SJ, Essigmann
JM (1990) Characterization of a DNA damage-recognition protein from mammalian
cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the
anticancer drug cisplatin. Biochemistry 29:5872-5880
53.
Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M, Samudio I, Kladde MP,
Vyhlidal C, Safe S (1999) Mechanisms of transcriptional activation of bcl-2 gene
expression by 17beta-estradiol in breast cancer cells. J Biol Chem 274:3209932107
54.
Driscoll MD, Sathya G, Muyan M, Klinge CM, Hilf R, Bambara RA (1998)
Sequence requirements for estrogen receptor binding to estrogen response
elements. J Biol Chem 273:29321-29330
55.
du Bois A, Neijt JP, Thigpen JT (1999) First line chemotherapy with carboplatin
plus paclitaxel in advanced ovarian cancer--a new standard of care? Ann Oncol 10
Suppl 1:35-41
56.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001)
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian
cells. Nature 411:494-498
243
57. Essigmann JM, Rink SM, Park HJ, Croy RG (2001b) Design of DNA damaging
agents that hijack transcription factors and block DNA repair. Adv Exp Med Biol
500:301-313
58.
Essigmann, Rink SM, Park HJ, Croy RG (2001a) Design of DNA damaging agents
that hijack transcription factors and block DNA repair. In: Dansette (ed) Biological
Reactive Intermediates VI, 6 edn
59.
Festjens N,Vanden BT, Vandenabeele P (2006) Necrosis, a well-orchestrated
form of cell demise: signalling cascades, important mediators and concomitant
immune response. Biochim Biophys Acta 1757:1371-1387
60. Fliss AE, Benzeno S, Rao J, Caplan AJ (2000) Control of estrogen receptor ligand
binding by Hsp90. J Steroid Biochem Mol Biol 72:223-230
61.
Foulds (1954) The experimental study of tumor progression: a review. Cancer Res
14:327-339
62.
Friedberg EC, Bonura T, Love JD, McMillan S, Radany EH, Schultz RA (1981) The
repair of DNA damage: recent developments and new insights. J Supramol Struct
Cell Biochern 16:91-103
-
63.
Friedberg EC, Walker G,Siede W (1995) DNA repair and mutagenesis
64.
Fu XD, Simoncini T (2008) Extra-nuclear signaling of estrogen receptors. IUBMB
Life 60:502-510
244
65.
Furuta T, Ueda T, Aune G, Sarasin A, Kraemer KH, Pommier Y (2002)
Transcription-coupled nucleotide excision repair as a determinant of cisplatin
sensitivity of human cells. Cancer Res 62:4899-4902
66.
Gibb RK, Taylor DD, Wan T, O'Connor DM, Doering DL, Gercel-Taylor C (1997)
Apoptosis as a measure of chemosensitivity to cisplatin and taxol therapy in
ovarian cancer cell lines. Gynecol Oncol 65:13-22
67.
Gozuacik D, Kimchi A (2007) Autophagy and cell death. Curr Top Dev Biol 78:217245
68.
Grant DF, Bessho T, Reardon JT (1998) Nucleotide excision repair of melphalan
monoadducts. Cancer Res 58:5196-5200
69.
Graves PR, Yu L, Schwarz JK, Gales J, Sausville EA, O'Connor PM, PiwnicaWorms H (2000) The Chk1 protein kinase and the Cdc25C regulatory pathways
are targets of the anticancer agent UCN-01. J Biol Chem 275:5600-5605
70. Green S, Walter P, Greene G, Krust A, Goffin C, Jensen E, Scrace G, Waterfield M,
Chambon P (1986) Cloning of the human oestrogen receptor cDNA. J Steroid
Biochem 24:77-83
71.
Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J (1986) Sequence and
expression of human estrogen receptor complementary DNA. Science 231:11501154
72.
Greenlee RT, Hill-Harmon MB, Murray T, Thun M (2001) Cancer statistics, 2001.
CA Cancer J Clin 51:15-36
245
73. Greenlee RT, Murray T, Bolden S, Wingo PA (2000) Cancer statistics, 2000. CA
Cancer J Clin 50:7-33
74. Haapala E, Hakala K, Jokipelto E, Vilpo J, Hovinen J (2001) Reactions of N,Nbis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'deoxyguanosine. Chem Res Toxicol 14:988-995
75.
Hagopian GS, Mills GB, Khokhar AR, Bast RC, Jr., Siddik ZH (1999) Expression of
p53 in cisplatin-resistant ovarian cancer cell lines: modulation with the novel
platinum analogue (1R,2R-diaminocyclohexane)(trans-diacetato)(dichloro)platinum(IV). Clin Cancer Res 5:655-663
76.
Hall JM, McDonnell DP (1999) The estrogen receptor beta-isoform (ERbeta) of the
human estrogen receptor modulates ERalpha transcriptional activity and is a key
regulator of the cellular response to estrogens and antiestrogens. Endocrinology
140:5566-5578
77. Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, WhangPeng J, Rogan AM, Green WR, Ozols RF (1983) Characterization of a human
ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors.
Cancer Res 43:5379-5389
78. Hamilton TC, Young RC, Ozols RF (1984) Experimental model systems of ovarian
cancer: applications to the design and evaluation of new treatment approaches.
Semin Oncol 11:285-298
79.
Hanahan D,Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70
246
80. Hartley JA, Berardini MD, Souhami RL (1991) An agarose gel method for the
determination of DNA interstrand crosslinking applicable to the measurement of the
rate of total and "second-arm" crosslink reactions. Anal Biochem 193:131-134
81.
Havrilesky U, McMahon CP, Lobenhofer EK, Whitaker R, Marks JR, Berchuck A
(2001) Relationship between expression of coactivators and corepressors of
hormone receptors and resistance of ovarian cancers to growth regulation by
steroid hormones. J Soc Gynecol Investig 8:104-113
82. He Z, Ingles CJ (1997) Isolation of human complexes proficient in nucleotide
excision repair. Nucleic Acids Res 25:1136-1141
83.
Helleday T, Petermann E, Lundin C, Hodgson B, Sharma RA (2008) DNA repair
pathways as targets for cancer therapy. Nat Rev Cancer 8:193-204
84.
Hippert MM, O'Toole PS, Thorburn A (2006) Autophagy in cancer: good, bad, or
both? Cancer Res 66:9349-9351
85.
Ho SM (2003) Estrogen, progesterone and epithelial ovarian cancer. Reprod Biol
Endocrinol 1:73
86.
Htun H, Holth LT, Walker D, Davie JR, Hager GL (1999) Direct visualization of the
human estrogen receptor alpha reveals a role for ligand in the nuclear distribution
of the receptor. Mol Biol Cell 10:471-486
87. Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995d) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
247
88. Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995a) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
89. Hua W, Christianson T, Rougeot C, Rochefort H, Clinton GM (1995b) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
90. Hua W, Christianson T, Rougeot C, Rochefort H,Clinton GM (1995c) SKOV3
ovarian carcinoma cells have functional estrogen receptor but are growth-resistant
to estrogen and antiestrogens. J Steroid Biochem Mol Biol 55:279-289
91.
Huang A, Kaley G (2004) Gender-specific regulation of cardiovascular function:
estrogen as key player. Microcirculation 11:9-38
92.
Huang JC, Zamble DB, Reardon JT, Lippard SJ, Sancar A (1994) HMG-domain
proteins specifically inhibit the repair of the major DNA adduct of the anticancer
drug cisplatin by human excision nuclease. Proc NatI Acad Sci U S A 91:1039410398
93. Hurley LH (2002) DNA and its associated processes as targets for cancer therapy.
Nat Rev Cancer 2:188-200
94. Igney FH, Krammer PH (2002) Death and anti-death: tumour resistance to
apoptosis. Nat Rev Cancer 2:277-288
95. Jacobs I, Bast RC, Jr. (1989) The CA 125 tumour-associated antigen: a review of
the literature. Hum Reprod 4:1-12
248
96.
Jaffe N, Frei E, Ill, Traggis D, Bishop Y (1974) Adjuvant methotrexate and
citrovorum-factor treatment of osteogenic sarcoma. N Engl J Med 291:994-997
97.
Jakowlew SB, Breathnach R, Jeltsch JM, Masiakowski P, Chambon P (1984)
Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell
line MCF-7. Nucleic Acids Res 12:2861-2878
98.
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer
statistics, 2008. CA Cancer J Clin 58:71-96
99.
Jones CJ, Wood RD (1993) Preferential binding of the xeroderma pigmentosum
group A complementing protein to damaged DNA. Biochemistry 32:12096-12104
100. Kim DH, Rossi JJ (2007) Strategies for silencing human disease using RNA
interference. Nat Rev Genet 8:173-184
101. King WJ, Greene GL (1984) Monoclonal antibodies localize oestrogen receptor in
the nuclei of target cells. Nature 307:745-747
102. Klein-Hitpass L, Ryffel GU, Heitlinger E, Cato AC (1988) A 13 bp palindrome is a
functional estrogen responsive element and interacts specifically with estrogen
receptor. Nucleic Acids Res 16:647-663
103. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M,
Baehrecke EH, Bahr BA, Ballabio A, Bamber BA, Bassham DC, Bergamini E, Bi X,
Biard-Piechaczyk M, Blum JS, Bredesen DE, Brodsky JL, Brumell JH, Brunk UT,
Bursch W, Camougrand N, Cebollero E, Cecconi F, Chen Y, Chin LS, Choi A, Chu
CT, Chung J, Clarke PG, Clark RS, Clarke SG, Clave C, Cleveland JL, Codogno P,
Colombo MI, Coto-Montes A, Cregg JM, Cuervo AM, Debnath J, Demarchi F,
249
(%
Dennis PB, Dennis PA, Deretic V, Devenish RJ, Di SF, Dice JF, Difiglia M, neshKumar S, Distelhorst CW, Djavaheri-Mergny M, Dorsey FC, Droge W, Dron M,
Dunn WA, Jr., Duszenko M, Eissa NT, Elazar Z, Esclatine A, Eskelinen EL, Fesus
L, Finley KD, Fuentes JM, Fueyo J, Fujisaki K, Galliot B, Gao FB, Gewirtz DA,
Gibson SB, Gohla A, Goldberg AL, Gonzalez R, Gonzalez-Estevez C, Gorski S,
Gottlieb RA, Haussinger D, He YW, Heidenreich K, Hill JA, Hoyer-Hansen M, Hu X,
Huang WP, Iwasaki A, Jaattela M, Jackson WT, Jiang X, Jin S, Johansen T, Jung
JU, Kadowaki M, Kang C, Kelekar A, Kessel DH, Kiel JA, Kim HP, Kimchi A,
Kinsella TJ, Kiselyov K, Kitamoto K, Knecht E, Komatsu M, Kominami E, Kondo S,
Kovacs AL, Kroemer G, Kuan CY, Kumar R, Kundu M, Landry J, Laporte M, Le W,
Lei HY, Lenardo MJ, Levine B, Lieberman A, Lim KL, Lin FC, Liou W, Liu LF,
Lopez-Berestein G, Lopez-Otin C, Lu B, Macleod KF, Malorni W, Martinet W,
Matsuoka K, Mautner J, Meijer AJ, Melendez A, Michels P, Miotto G, Mistiaen WP,
Mizushima N, Mograbi B, Monastyrska I, Moore MN, Moreira PI, Moriyasu Y, Motyl
T, Munz C, Murphy LO, Naqvi NI, Neufeld TP, Nishino I, Nixon RA, Noda T,
Nurnberg B, Ogawa M, Oleinick NL, Olsen LJ, Ozpolat B, Paglin S, Palmer GE,
Papassideri I, Parkes M, Perlmutter DH, Perry G, Piacentini M, Pinkas-Kramarski
R, Prescott M, Proikas-Cezanne T, Raben N, Rami A, Reggiori F, Rohrer B,
Rubinsztein DC, Ryan KM, Sadoshima J, Sakagami H, Sakai Y, Sandri M,
Sasakawa C, Sass M, Schneider C, Seglen PO, Seleverstov 0, Settleman J,
Shacka JJ, Shapiro IM, Sibirny A, Silva-Zacarin EC, Simon HU, Simone C,
Simonsen A, Smith MA, Spanel-Borowski K, Srinivas V, Steeves M, Stenmark H,
Stromhaug PE, Subauste CS, Sugimoto S, Sulzer D, Suzuki T, Swanson MS,
Tabas I, Takeshita F, Talbot NJ, Talloczy Z, Tanaka K, Tanaka K, Tanida 1,Taylor
GS, Taylor JP, Terman A, Tettamanti G, Thompson CB, Thumm M, Tolkovsky AM,
Tooze SA, Truant R, Tumanovska LV, Uchiyama Y, Ueno T, Uzcategui NL, van dK,
250
I, Vaquero EC, Vellai T, Vogel MW, Wang HG, Webster P, Wiley JW, Xi Z, Xiao G,
Yahalom J, Yang JM, Yap G, Yin XM, Yoshimori T, Yu L,Yue Z, Yuzaki M,
Zabirnyk 0, Zheng X, Zhu X, Deter RL (2008) Guidelines for the use and
interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy
4:151-175
104. Koberle B, Roginskaya V, Wood RD (2006) XPA protein as a limiting factor for
nucleotide excision repair and UV sensitivity in human cells. DNA Repair (Amst)
5:641-648
105. Kohn KW (1996) Beyond DNA cross-linking: history and prospects of DNAtargeted cancer treatment--fifteenth Bruce F. Cain Memorial Award Lecture.
Cancer Res 56:5533-5546
106. Kohn KW, Hartley JA, Mattes WB (1987) Mechanisms of DNA sequence selective
alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res
15:10531-10549
107. Kolfschoten GM, Hulscher TM, Pinedo HM, Boven E (2000) Drug resistance
features and S-phase fraction as possible determinants for drug response in a
panel of human ovarian cancer xenografts. Br J Cancer 83:921-927
108. Krasner C (2007) Aromatase inhibitors in gynecologic cancers. J Steroid Biochem
Mol Biol 106:76-80
109. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson
JA (1997) Comparison of the ligand binding specificity and transcript tissue
distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870
251
110. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996) Cloning of
a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A
93:5925-5930
111. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van
der BB, Gustafsson JA (1998a) Interaction of estrogenic chemicals and
phytoestrogens with estrogen receptor beta. Endocrinology 139:4252-4263
112. Kuiper GG, Shughrue PJ, Merchenthaler I, Gustafsson JA (1998b) The estrogen
receptor beta subtype: a novel mediator of estrogen action in neuroendocrine
systems. Front Neuroendocrinol 19:253-286
113. Kumar R,Thompson EB (1999) The structure of the nuclear hormone receptors.
Steroids 64:310-319
114. Kundu GC, Schullek JR, Wilson IB(1994) The alkylating properties of chlorambucil.
Pharmacol Biochem Behav 49:621-624
115. Kurreck J (2009) RNA interference: from basic research to therapeutic applications.
Angew Chem Int Ed Engl 48:1378-1398
116. Kushner PJ, Agard D, Feng WJ, Lopez G, Schiau A, Uht R,Webb P, Greene G
(2000a) Oestrogen receptor function at classical and alternative response
elements. Novartis Found Symp 230:20-26
117. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P
(2000b) Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311317
252
118. Lahooti H, White R, Danielian PS, Parker MG (1994) Characterization of liganddependent phosphorylation of the estrogen receptor. Mol Endocrinol 8:182-188
119. Langdon SP, Hirst GL, Miller EP, Hawkins RA, Tesdale AL, Smyth JF, Miller WR
(1994) The regulation of growth and protein expression by estrogen in vitro: a
study of 8 human ovarian carcinoma cell lines. J Steroid Biochem Mol Biol 50:131135
120. Langdon SP, Ritchie A, Young K, Crew AJ, Sweeting V, Bramley T, Hillier S,
Hawkins RA, Tesdale AL, Smyth JF, . (1993) Contrasting effects of 17 betaestradiol on the growth of human ovarian carcinoma cells in vitro and in vivo. Int J
Cancer 55:459-464
121. Lau CC, Pardee AB (1982) Mechanism by which caffeine potentiates lethality of
nitrogen mustard. Proc Natl Acad Sci U S A 79:2942-2946
122. Lau KM, Mok SC, Ho SM (1999) Expression of human estrogen receptor-alpha
and -beta, progesterone receptor, and androgen receptor mRNA in normal and
malignant ovarian epithelial cells. Proc Natl Acad Sci U S A 96:5722-5727
123. Lawley PD (1966) Effects of some chemical mutagens and carcinogens on nucleic
acids. Prog Nucleic Acid Res Mol Biol 5:89-131
124. Lazennec G (2006) Estrogen receptor beta, a possible tumor suppressor involved
in ovarian carcinogenesis. Cancer Lett 231:151-157
125. Le GP, Montano MM, Schodin DJ, Katzenellenbogen BS (1994) Phosphorylation of
the human estrogen receptor. Identification of hormone-regulated sites and
253
examination of their influence on transcriptional activity. J Biol Chem 269:44584466
126. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular
mechanisms and biological functions of autophagy. Dev Cell 6:463-477
127. Li M, HERTZ R,BERGENSTAL DM (1958) Therapy of choriocarcinoma and
related trophoblastic tumors with folic acid and purine antagonists. N Engl J Med
259:66-74
128. Liberman RG, Tannenbaum SR, Hughey BJ, Shefer RE, Klinkowstein RE, Prakash
C, Harriman SP, Skipper PL (2004) An interface for direct analysis of (14)c in
nonvolatile samples by accelerator mass spectrometry. Anal Chem 76:328-334
129. Lindahl T (1979) DNA glycosylases, endonucleases for apurinic/apyrimidinic sites,
and base excision-repair. Prog Nucleic Acid Res Mol Biol 22:135-192
130. Lindgren PR, Cajander S, Backstrom T, Gustafsson JA, Makela S, Olofsson JI
(2004) Estrogen and progesterone receptors in ovarian epithelial tumors. Mol Cell
Endocrinol 221:97-104
131. Lindor NM, Petersen GM, Hadley DW, Kinney AY, Miesfeldt S, Lu KH, Lynch P,
Burke W, Press N (2006) Recommendations for the care of individuals with an
inherited predisposition to Lynch syndrome: a systematic review. JAMA 296:15071517
132. Lukanova A, Kaaks R (2005) Endogenous hormones and ovarian cancer:
epidemiology and current hypotheses. Cancer Epidemiol Biomarkers Prev 14:98107
254
133. MacLeod MC, Powell KL, Tran N (1995) Binding of the transcription factor, Spl, to
non-target sites in DNA modified by benzo[a]pyrene diol epoxide. Carcinogenesis
16:975-983
134. Maggi A, Ciana P, Belcredito S, Vegeto E (2004) Estrogens in the nervous system:
mechanisms and nonreproductive functions. Annu Rev Physiol 66:291-313
135. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K,
Blumberg B, Kastner P, Mark M, Chambon P, Evans RM (1995) The nuclear
receptor superfamily: the second decade. Cell 83:835-839
136. Masiakowski P, Breathnach R, Bloch J, Gannon F, Krust A, Chambon P (1982)
Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human
breast cancer cell line. Nucleic Acids Res 10:7895-7903
137. Masood S, Heitmann J, Nuss RC, Benrubi GI (1989) Clinical correlation of
hormone receptor status in epithelial ovarian cancer. Gynecol Oncol 34:57-60
138. May FE, Westley BR (1988) Identification and characterization of estrogenregulated RNAs in human breast cancer cells. J Biol Chem 263:12901-12908
139. May FE, Westley BR (1995) Estrogen regulated messenger RNAs in human breast
cancer cells. Biomed Pharmacother 49:400-414
140. McA'Nulty MM, Lippard SJ (1996) The HMG-domain protein Ixr1 blocks excision
repair of cisplatin-DNA adducts in yeast. Mutat Res 362:75-86
255
141. McA'Nulty MM, Whitehead JP, Lippard SJ (1996) Binding of lxrl, a yeast HMGdomain protein, to cisplatin-DNA adducts in vitro and in vivo. Biochemistry
35:6089-6099
142. Meijer AJ, Codogno P (2006) Signalling and autophagy regulation in health, aging
and disease. Mol Aspects Med 27:411-425
143. Merajver SD, Pham TM, Caduff RF, Chen M, Poy EL, Cooney KA, Weber BL,
Collins FS, Johnston C, Frank TS (1995) Somatic mutations in the BRCA1 gene in
sporadic ovarian tumours. Nat Genet 9:439-443
144. Meyn, Murray D (1986) Cell cycle Effects of alkylating agents.
145. Miki Y, Swensen J,Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu
Q, Cochran C, Bennett LM, Ding W, . (1994) A strong candidate for the breast and
ovarian cancer susceptibility gene BRCA1. Science 266:66-71
146. Mitra K, Marquis JC, Hillier SM, Rye PT, Zayas B, Lee AS, Essigmann JM, Croy
RG (2002) A rationally designed genotoxin that selectively destroys estrogen
receptor-positive breast cancer cells. J Am Chem Soc 124:1862-1863
147. Mizushima N (2007) Autophagy: process and function. Genes Dev 21:2861-2873
148. Mizushima N, Levine B,Cuervo AM, Klionsky DJ (2008) Autophagy fights disease
through cellular self-digestion. Nature 451:1069-1075
149. Moretti-Rojas I, Fuqua SA, Montgomery RA, Ill, McGuire WL (1988) A cDNA for
the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells. Breast
Cancer Res Treat 11:155-163
256
150. Morimoto H, Safrit JT, Bonavida B (1991) Synergistic effect of tumor necrosis
factor-alpha- and diphtheria toxin-mediated cytotoxicity in sensitive and resistant
human ovarian tumor cell lines. J Immunol 147:2609-2616
151. Morimoto H,Yonehara S, Bonavida B (1993) Overcoming tumor necrosis factor
and drug resistance of human tumor cell lines by combination treatment with antiFas antibody and drugs or toxins. Cancer Res 53:2591-2596
152. Morisset M, Capony F, Rochefort H (1986) Processing and estrogen regulation of
the 52-kilodalton protein inside MCF7 breast cancer cells. Endocrinology
119:2773-2782
153. Morissette M, Le SM, D'Astous M, Jourdain S, Al SS, Morin N, Estrada-Camarena
E, Mendez P, Garcia-Segura LM, Di PT (2008) Contribution of estrogen receptors
alpha and beta to the effects of estradiol in the brain. J Steroid Biochem Mol Biol
108:327-338
154. Mutch DG (2002) Surgical management of ovarian cancer. Semin Oncol 29:3-8
155. Narod SA, Foulkes WD (2004) BRCA1 and BRCA2: 1994 and beyond. Nat Rev
Cancer 4:665-676
156. Nash JD, Ozols RF, Smyth JF, Hamilton TC (1989) Estrogen and anti-estrogen
effects on the growth of human epithelial ovarian cancer in vitro. Obstet Gynecol
73:1009-1016
157. Neijt JP, Engelholm SA, Tuxen MK, Sorensen PG, Hansen M, Sessa C, de Swart
CA, Hirsch FR, Lund B,van Houwelingen HC (2000) Exploratory phase Ill study of
257
paclitaxel and cisplatin versus paclitaxel and carboplatin in advanced ovarian
cancer. J Clin Oncol 18:3084-3092
158. Newton K, Strasser A (1998) The BcI-2 family and cell death regulation. Curr Opin
Genet Dev 8:68-75
159. Nguyen HN, Sevin BU, Averette HE, Perras J, Ramos R, Donato D,Ochiai K,
Penalver M (1993) Cell cycle perturbations of platinum derivatives on two ovarian
cancer cell lines. Cancer Invest 11:264-275
160. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J,Andersson G, Enmark E,
Pettersson K,Warner M, Gustafsson JA (2001) Mechanisms of estrogen action.
Physiol Rev 81:1535-1565
161. Noy N (2007) Ligand specificity of nuclear hormone receptors: sifting through
promiscuity. Biochemistry 46:13461-13467
162. O'Connor PM, Kohn KW (1990) Comparative pharmacokinetics of DNA lesion
formation and removal following treatment of LI 210 cells with nitrogen mustards.
Cancer Commun 2:387-394
163. O'Donnell AJ, Macleod KG, Burns DJ, Smyth JF, Langdon SP (2005) Estrogen
receptor-alpha mediates gene expression changes and growth response in ovarian
cancer cells exposed to estrogen. Endocr Relat Cancer 12:851-866
164. Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, Muramatsu M
(1998) The complete primary structure of human estrogen receptor beta (hER beta)
and its heterodimerization with ER alpha in vivo and in vitro. Biochem Biophys Res
Commun 243:122-126
258
165. Olefsky JM (2001) Nuclear receptor minireview series. J Biol Chem 276:3686336864
166. Olive PL, Banath JP (1995) Sizing highly fragmented DNA in individual apoptotic
cells using the comet assay and a DNA crosslinking agent. Exp Cell Res 221:19-26
167. Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage
in individual cells. Nat Protoc 1:23-29
168. Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA
damage and repair in tumor and normal cells measured using the "comet" assay.
Radiat Res 122:86-94
169. Osborne CK, Yochmowitz MG, Knight WA, Ill, McGuire WL (1980) The value of
estrogen and progesterone receptors in the treatment of breast cancer. Cancer
46:2884-2888
170. Oxelmark E, Knoblauch R, Arnal S, Su LF, Schapira M, Garabedian MJ (2003)
Genetic dissection of p23, an Hsp9O cochaperone, reveals a distinct surface
involved in estrogen receptor signaling. J Biol Chem 278:36547-36555
171. Ozols RF (2005) Treatment goals in ovarian cancer. Int J Gynecol Cancer 15
Suppl 1:3-11
172. Ozols RF, Bundy BN, Greer BE, Fowler JM, Clarke-Pearson D, Burger RA, Mannel
RS, DeGeest K, Hartenbach EM, Baergen R (2003) Phase Ill trial of carboplatin
and paclitaxel compared with cisplatin and paclitaxel in patients with optimally
resected stage Ill ovarian cancer: a Gynecologic Oncology Group study. J Clin
Oncol 21:3194-3200
259
173. Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, Domingo D,
Yahalom J (2001) A novel response of cancer cells to radiation involves autophagy
and formation of acidic vesicles. Cancer Res 61:439-444
174. Pal T, Permuth-Wey J,Betts JA, Krischer JP, Fiorica J, Arango H, LaPolla J,
Hoffman M, Martino MA, Wakeley K,Wilbanks G, Nicosia S, Cantor A, Sutphen R
(2005) BRCA1 and BRCA2 mutations account for a large proportion of ovarian
carcinoma cases. Cancer 104:2807-2816
175. Papac RJ (2001) Origins of cancer therapy. Yale J Biol Med 74:391-398
176. Parker MG (1991) Nuclear Hormone Receptors
177. Parker, Jensen EV (1991) Overview of the Nuclear Receptor Family.
178. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA
Cancer J Clin 55:74-108
179. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M,
Schneider MD, Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1dependent autophagy. Cell 122:927-939
180. Pearce ST, Jordan VC (2004) The biological role of estrogen receptors alpha and
beta in cancer. Crit Rev Oncol Hematol 50:3-22
181. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA (1997) Mouse estrogen
receptor beta forms estrogen response element-binding heterodimers with
estrogen receptor alpha. Mol Endocrinol 11:1486-1496
260
182. Piccart MJ, du BA, Gore ME, Neijt JP, Pecorelli S, Pujade-Lauraine E (2000) A
new standard of care for treatment of ovarian cancer. Eur J Cancer 36:10-12
183. Pil PM, Lippard SJ (1992) Specific binding of chromosomal protein HMG1 to DNA
damaged by the anticancer drug cisplatin. Science 256:234-237
184. Pratt WB, Ruddon RW, Ensminger WD, Maybaum J (1994) The Anticancer drugs
185. Pujol P, Rey JM, Nirde P, Roger P, Gastaldi M, Laffargue F, Rochefort H,
Maudelonde T (1998) Differential expression of estrogen receptor-alpha and -beta
messenger RNAs as a potential marker of ovarian carcinogenesis. Cancer Res
58:5367-5373
186. Rao BR, Slotman BJ (1991) Endocrine factors in common epithelial ovarian cancer.
Endocr Rev 12:14-26
187. Rao GG, Miller DS (2005) Clinical applications of hormonal therapy in ovarian
cancer. Curr Treat Options Oncol 6:97-102
188. Razandi M, Pedram A, Merchenthaler 1,Greene GL, Levin ER (2004) Plasma
membrane estrogen receptors exist and functions as dimers. Mol Endocrinol
18:2854-2865
189. Reardon JT, Vaisman A, Chaney SG, Sancar A (1999) Efficient nucleotide excision
repair of cisplatin, oxaliplatin, and Bis-aceto-ammine-dichloro-cyclohexylamineplatinum(IV) (JM216) platinum intrastrand DNA diadducts. Cancer Res 59:39683971
261
190. Reed (2006) Cisplatin, carboplatin and oxaliplatin. In: Chabner BA, Longo DL (eds)
Cancer chemotherapy and biotherapy, 4 edn
191. Reggiori F, Klionsky DJ (2002) Autophagy in the eukaryotic cell. Eukaryot Cell
1:11-21
192. Renan MJ (1993) How many mutations are required for tumorigenesis?
Implications from human cancer data. Mol Carcinog 7:139-146
193. Reynolds A, Anderson EM, Vermeulen A, Fedorov Y, Robinson K, Leake D,
Karpilow J, Marshall WS, Khvorova A (2006) Induction of the interferon response
by siRNA is cell type- and duplex length-dependent. RNA 12:988-993
194. Riggs BL, Khosla S, Melton U, Ill (2002) Sex steroids and the construction and
conservation of the adult skeleton. Endocr Rev 23:279-302
195. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2001) Risk factors for epithelial borderline ovarian tumors: results of a
Swedish case-control study. Gynecol Oncol 83:575-585
196. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Persson IR (2002a) Risk factors for invasive epithelial ovarian cancer: results from
a Swedish case-control study. Am J Epidemiol 156:363-373
197. Riman T, Dickman PW, Nilsson S, Correia N, Nordlinder H, Magnusson CM,
Weiderpass E, Persson IR (2002b) Hormone replacement therapy and the risk of
invasive epithelial ovarian cancer in Swedish women. J Natl Cancer Inst 94:497504
262
198. Riman T, Dickman PW, Nilsson S, Nordlinder H, Magnusson CM, Persson IR
(2004) Some life-style factors and the risk of invasive epithelial ovarian cancer in
Swedish women. Eur J Epidemiol 19:1011-1019
199. Rimer, Schildkraut JM (1997) Cancer Screening. In: DeVita VT, Hellman S,
Rosenberg SA (eds), Fifth edn pp 619-631
200. Rink SM, Yarema KJ, Solomon MS, Paige LA, Tadayoni-Rebek BM, Essigmann
JM, Croy RG (1996) Synthesis and biological activity of DNA damaging agents that
form decoy binding sites for the estrogen receptor. Proc Nati Acad Sci U S A
93:15063-15068
201. Risch HA (2002) Hormone replacement therapy and the risk of ovarian cancer.
Gynecol Oncol 86:115-117
202. Roberts D, Schick J, Conway S, Biade S, Laub PB, Stevenson JP, Hamilton TC,
O'Dwyer PJ, Johnson SW (2005) Identification of genes associated with platinum
drug sensitivity and resistance in human ovarian cancer cells. Br J Cancer
92:1149-1158
203. Rochefort H (1990) Cathepsin D in breast cancer. Breast Cancer Res Treat 16:313
204. Rochefort H (1998) [Estrogens, cathepsin D and metastasis in cancers of the
breast and ovary: invasion or proliferation?]. C R Seances Soc Biol Fil 192:241-251
205. Rochefort H (1999) [Estrogen-induced genes in breast cancer, and their medical
importance]. Bull Acad Natl Med 183:955-968
263
206. Rose PG, Reale FR, Longcope C, Hunter RE (1990) Prognostic significance of
estrogen and progesterone receptors in epithelial ovarian cancer. Obstet Gynecol
76:258-263
207. Roy G, Horton JK, Roy R, Denning T, Mitra S, Boldogh I (2000) Acquired alkylating
drug resistance of a human ovarian carcinoma cell line is unaffected by altered
levels of pro- and anti-apoptotic proteins. Oncogene 19:141-150
208. Russo IH, Russo J (1998) Role of hormones in mammary cancer initiation and
progression. J Mammary Gland Biol Neoplasia 3:49-61
209. Rutherford T, Brown WD, Sapi E, Aschkenazi S, Munoz A, Mor G (2000) Absence
of estrogen receptor-beta expression in metastatic ovarian cancer. Obstet Gynecol
96:417-421
210. Satokata 1,Tanaka K, Miura N, Narita M, Mimaki T, Satoh Y, Kondo S, Okada Y
(1992) Three nonsense mutations responsible for group A xeroderma
pigmentosum. Mutat Res 273:193-202
211. Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA,
Safe S (2000) Ligand-, cell-, and estrogen receptor subtype (alpha/beta)dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:53795387
212. Sawyers C (2004) Targeted cancer therapy. Nature 432:294-297
213. Schaner ME, Ross DT, Ciaravino G, Sorlie T, Troyanskaya 0, Diehn M,Wang YC,
Duran GE, Sikic TL, Caldeira S, Skomedal H, Tu IP,Hernandez-Boussard T,
Johnson SW, O'Dwyer PJ, Fero MJ, Kristensen GB, Borresen-Dale AL, Hastie T,
264
Tibshirani R, van de RM, Teng NN, Longacre TA, Botstein D, Brown PO, Sikic BI
(2003) Gene expression patterns in ovarian carcinomas. Mol Biol Cell 14:43764386
214. Schildkraut JM, Schwingl PJ, Bastos E, Evanoff A, Hughes C (1996) Epithelial
ovarian cancer risk among women with polycystic ovary syndrome. Obstet Gynecol
88:554-559
215. Schlegel R, Pardee AB (1986) Caffeine-induced uncoupling of mitosis from the
completion of DNA replication in mammalian cells. Science 232:1264-1266
216. Schuchard M, Landers JP, Sandhu NP, Spelsberg TC (1993) Steroid hormone
regulation of nuclear proto-oncogenes. Endocr Rev 14:659-669
217. Segnitz B, Gehring U (1995) Subunit structure of the nonactivated human estrogen
receptor. Proc Natl Acad Sci U S A 92:2179-2183
218. Segnitz B, Gehring U (1997) The function of steroid hormone receptors is inhibited
by the hsp90-specific compound geldanamycin. J Biol Chem 272:18694-18701
219. Sekiguchi 1,Suzuki M, Tamada T, Shinomiya N, Tsuru S, Murata M (1996) Effects
of cisplatin on cell cycle kinetics, morphological change, and cleavage pattern of
DNA in two human ovarian carcinoma cell lines. Oncology 53:19-26
220. Serova M, Calvo F, Lokiec F, Koeppel F, Poindessous V, Larsen AK, Laar ES,
Waters SJ, Cvitkovic E, Raymond E (2006) Characterizations of irofulven
cytotoxicity in combination with cisplatin and oxaliplatin in human colon, breast,
and ovarian cancer cells. Cancer Chemother Pharmacol 57:491-499
265
221. Shapiro GI, Harper JW (1999) Anticancer drug targets: cell cycle and checkpoint
control. J Clin Invest 104:1645-1653
222. Sharma U, Marquis JC, Nicole DA, Hillier SM, Fedeles B, Rye PT, Essigmann JM,
Croy RG (2004) Design, synthesis, and evaluation of estradiol-linked genotoxicants
as anti-cancer agents. Bioorg Med Chem Lett 14:3829-3833
223. Shuey DJ, McCallus DE, Giordano T (2002) RNAi: gene-silencing in therapeutic
intervention. Drug Discov Today 7:1040-1046
224. Slotman BJ, Rao BR (1988) Ovarian cancer (review). Etiology, diagnosis,
prognosis, surgery, radiotherapy, chemotherapy and endocrine therapy. Anticancer
Res 8:417-434
225. States JC, McDuffie ER, Myrand SP, McDowell M, Cleaver JE (1998) Distribution
of mutations in the human xeroderma pigmentosum group A gene and their
relationships to the functional regions of the DNA damage recognition protein. Hum
Mutat 12:103-113
226. Stenoien DL, Mancini MG, Patel K,Allegretto EA, Smith CL, Mancini MA (2000)
Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1.
Mol Endocrinol 14:518-534
227. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, Mancini
MA (2001) FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and
proteasome-dependent. Nat Cell Biol 3:15-23
266
228. Stevens EV, Nishizuka S, Antony S, Reimers M, Varma S, Young L, Munson PJ,
Weinstein JN, Kohn EC, Pommier Y (2008) Predicting cisplatin and trabectedin
drug sensitivity in ovarian and colon cancers. Mol Cancer Ther 7:10-18
229. Sugiyama K, Shimizu M, Akiyama T, Tamaoki T, Yamaguchi K, Takahashi R,
Eastman A, Akinaga S (2000) UCN-01 selectively enhances mitomycin C
cytotoxicity in p53 defective cells which is mediated through S and/or G(2)
checkpoint abrogation. Int J Cancer 85:703-709
230. Sunters A, Springer CJ, Bagshawe KD, Souhami RL, Hartley JA (1992) The
cytotoxicity, DNA crosslinking ability and DNA sequence selectivity of the aniline
mustards melphalan, chlorambucil and 4-[bis(2-chloroethyl)amino] benzoic acid.
Biochem Pharmacol 44:59-64
231. Tew, Colvin OM, Jones RB (2006) Clinical and High-Dose Alkylating Agents. In:
Chabner BA, Longo DL (eds) Cancer Chemotherapy and Biotherapy, 4 edn
232. Treiber DK, Zhai X, Jantzen HM, Essigmann JM (1994) Cisplatin-DNA adducts are
molecular decoys for the ribosomal RNA transcription factor hUBF (human
upstream binding factor). Proc Natl Acad Sci U S A 91:5672-5676
233. Turteltaub KW, Dingley KH (1998) Application of accelerated mass spectrometry
(AMS) in DNA adduct quantification and identification. Toxicol Lett 102-103:435439
234. Turteltaub KW, Felton JS, Gledhill BL, Vogel JS, Southon JR, Caffee MW, Finkel
RC, Nelson DE, Proctor ID, Davis JC (1990) Accelerator mass spectrometry in
biomedical dosimetry: relationship between low-level exposure and covalent
267
binding of heterocyclic amine carcinogens to DNA. Proc Natl Acad Sci U S A
87:5288-5292
235. Tyagi A, Singh RP, Agarwal C, Siriwardana S, Sclafani RA, Agarwal R (2005)
Resveratrol causes Cdc2-tyrl 5 phosphorylation via ATM/ATR-Chk 1/2-Cdc25C
pathway as a central mechanism for S phase arrest in human ovarian carcinoma
Ovcar-3 cells. Carcinogenesis 26:1978-1987
236. Vandenabeele P, Vanden BT, Festjens N (2006) Caspase inhibitors promote
alternative cell death pathways. Sci STKE 2006:e44
237. Venkitaraman AR (2004) Tracing the network connecting BRCA and Fanconi
anaemia proteins. Nat Rev Cancer 4:266-276
238. von Angerer E, Prekajac J, Strohmeier J (1984) 2-Phenylindoles. Relationship
between structure, estrogen receptor affinity, and mammary tumor inhibiting
activity in the rat. J Med Chem 27:1439-1447
239. Waldman T, Lengauer C, Kinzler KW, Vogelstein B (1996) Uncoupling of S phase
and mitosis induced by anticancer agents in cells lacking p21. Nature 381:713-716
240. Walker G, MacLeod K, Williams AR, Cameron DA, Smyth JF, Langdon SP (2007)
Estrogen-regulated gene expression predicts response to endocrine therapy in
patients with ovarian cancer. Gynecol Oncol 106:461-468
241. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E,
Scrace G, Waterfield M, . (1985) Cloning of the human estrogen receptor cDNA.
Proc Natl Acad Sci U S A 82:7889-7893
268
242. Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O'Connor PM (1996) UCN01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53.
J Nati Cancer Inst 88:956-965
243. Webb P, Lopez GN, Uht RM, Kushner PJ (1995) Tamoxifen activation of the
estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9:443-456
244. Webb P, Nguyen P,Valentine C, Lopez GN, Kwok GR, McInerney E,
Katzenellenbogen BS, Enmark E, Gustafsson JA, Nilsson S, Kushner PJ (1999)
The estrogen receptor enhances AP-1 activity by two distinct mechanisms with
different requirements for receptor transactivation functions. Mol Endocrinol
13:1672-1685
245. Wilson TM, Ewel A, Duguid JR, Eble JN, Lescoe MK, Fishel R, Kelley MR (1995)
Differential cellular expression of the human MSH2 repair enzyme in small and
large intestine. Cancer Res 55:5146-5150
246. Wolf JK, Mills GB, Bazzet L, Bast RC, Jr., Roth JA, Gershenson DM (1999)
Adenovirus-mediated p53 growth inhibition of ovarian cancer cells is independent
of endogenous p53 status. Gynecol Oncol 75:261-266
247. Wu X, Fan W, Xu S, Zhou Y (2003) Sensitization to the cytotoxicity of cisplatin by
transfection with nucleotide excision repair gene xeroderma pigmentosun group A
antisense RNA in human lung adenocarcinoma cells. Clin Cancer Res 9:58745879
269
248. Xu Y, Traystman RJ, Hurn PD, Wang MM (2004) Membrane restraint of estrogen
receptor alpha enhances estrogen-dependent nuclear localization and genomic
function. Mol Endocrinol 18:86-96
249. Yap TA, Carden CP, Kaye SB (2009) Beyond chemotherapy: targeted therapies in
ovarian cancer. Nat Rev Cancer 9:167-181
250. Zakeri Z, Bursch W, Tenniswood M, Lockshin RA (1995) Cell death: programmed,
apoptosis, necrosis, or other? Cell Death Differ 2:87-96
251. Zang XP, Pento JT (2008) SiRNA inhibition of ER-alpha expression reduces KGFinduced proliferation of breast cancer cells. Anticancer Res 28:2733-2735
252. Zheng L, Annab LA, Afshari CA, Lee WH, Boyer TG (2001) BRCA1 mediates
ligand-independent transcriptional repression of the estrogen receptor. Proc Natl
Acad Sci U S A 98:9587-9592
253. Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in
perspective. Nature 408:433-439
270