Checkpoint Responses in Cancer Therapy

Checkpoint Responses
in Cancer Therapy
Cancer Drug Discovery
and Development™
Beverly A. Teicher, Series Editor
Checkpoint Responses in Cancer
Therapy, edited by Wei Dai, 2008
Cancer Proteomics: From Bench to
Bedside, edited by Sayed S. Daoud,
Antiangiogenic Agents in Cancer
Therapy, Second Edition, edited
by Beverly A. Teicher and Lee M.
Ellis, 2007
Apoptosis and Senescence in Cancer
Chemotherapy and
Radiotherapy, Second Edition,
edited by David A. Gerwitz, Shawn
Edan Holtz, and Steven Grant,
Molecular Targeting in Oncology,
edited by Howard L. Kaufman,
Scott Wadler, and Karen Antman,
In Vivo Imaging of Cancer Therapy,
edited by Anthony F. Shields and
Patricia Price, 2007
Transforming Growth Factor- in
Cancer Therapy, Volume II:
Cancer Treatment and Therapy,
edited by Sonia Jakowlew, 2008
Transforming Growth Factor- in
Cancer Therapy, Volume 1: Basic
and Clinical Biology, edited by
Sonia Jakowlew, 2008
Microtubule Targets in Cancer
Therapy, edited by Antonio T.
Fojo, 2007
Cytokines in the Genesis and
Treatment of Cancer, edited by
Michael A. Caligiuri, Michael T.
Lotze, and Frances R. Balkwill,
Regional Cancer Therapy, edited by
Peter M. Schlag and Ulrike Stein,
Gene Therapy for Cancer, edited by
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Vorburger, and Stephen G.
Swisher, 2007
Deoxynucleoside Analogs in Cancer
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Peters, 2006
Cancer Drug Resistance, edited by
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Histone Deacetylases: Transcriptional
Regulation and Other Cellular
Functions, edited by Eric Verdin,
Immunotherapy of Cancer, edited by
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Molecular Diagnostics for
Predicting and Monitoring
Therapeutic Effect, edited by
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Protein Tyrosine Kinases: From
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Bone Metastasis: Experimental and
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The Oncogenomics Handbook, edited
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Camptothecins in Cancer Therapy,
edited by Thomas G. Burke and
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Responses in
Cancer Therapy
Edited by
Wei Dai, phd
Department of Environmental Medicine,
New York University School of Medicine,
Tuxedo, NY
Wei Dai, PhD
Department of Environmental Medicine
New York University School of Medicine
Tuxedo, NY
Series Editor
Beverly A. Teicher, PhD
Genzyme Corporation
Framingham, MA
ISBN: 978-1-58829-930-7
e-ISBN: 978-1-59745-274-8
Library of Congress Control Number: 2007940763
©2008 Humana Press, a part of Springer Science+Business Media, LLC
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Cover illustration: Fig. 2A-C, Chapter 11, “Antiproliferation Inhibitors Targeting Aurora Kinases,”
by Kishore Shakalya and Daruka Mahadevan.
Printed on acid-free paper
The cell cycle is tightly regulated by a number of molecular entities
that maintain the genetic integrity of the cell and ensure that genetic
information is passed correctly to the daughter cells. Starting in the
1980s, extensive research efforts have revealed the existence of evolutionarily conserved surveillance mechanisms—commonly referred to
as checkpoints—that regulate the cell cycle by monitoring specific cellcycle processes and block progression through the cell cycle until these
processes have been completed accurately. If any of the molecular
events underlying these processes are impaired, the cell cycle stops to
allow the cell time for repair. If the cell is damaged beyond repair,
it will commit suicide by activating apoptotic processes. The loss
of checkpoint function often results in infidelity of DNA replication,
chromosome mis-segregation, or both, thereby predisposing the cell to
genetic instability and neoplastic transformation.
Cancer frequently results from damage to multiple genes controlling
cell division. Existing evidence indicates that cancer cells are
frequently defective in regulating one cell-cycle checkpoint, which
makes them very susceptible to insults to a second checkpoint, resulting
in apoptosis. Based on their mechanism of action, current antitumor
drugs that target the cell cycle can generally be divided into three
categories: inhibition of DNA synthesis, induction of DNA damage,
and disruption of mitotic processes. Most cancer drugs used in clinical
settings kill cancer cells by altering the DNA structure and inhibiting
DNA replication. Such events inevitably lead to activation of the
DNA replication or the DNA damage checkpoint. Since the 1980s,
much effort has also been directed to the discovery of mitotic targets
or processes, which, if altered, can lead to a mitotic catastrophe (a
specialized case of apoptosis). It is known that mitotic processes are
closely monitored by several surveillance mechanisms, including the
G2/M transition checkpoint, the prophase stress checkpoint, and the
spindle checkpoint. A defect in the regulation of any mitotic checkpoint often results in genomic instability, which predisposes the cell to
malignant transformation. By characterizing the molecular components
of cell-cycle checkpoint mechanisms and exploring differences in the
checkpoint status between normal cells and malignant cells, we may be
able to facilitate the discovery and development of chemotherapeutic
compounds that are more effective and more specific for tumor cells.
Checkpoint Responses in Cancer Therapy presents summaries of
the advances made since the 1980s in identifying various components
of cell-cycle checkpoints, elucidating their molecular regulation during
checkpoint activation, and validating the use of checkpoint proteins as
targets for the development of anticancer drugs. Proteins important for
G1 progression are known to mediate cell-cycle checkpoint responses,
especially after treatment with chemotherapeutic agents. Considerably
less is known about the G1 or G1/S checkpoint than other cell-cycle
checkpoints, in terms of their usefulness as targets for anticancer drug
discovery. We do know that the Rb protein (pRb), a major tumor
suppressor in the cells, plays a pivotal role in the negative control of
the cell cycle. The pRb is responsible for regulating the G1 checkpoint,
blocking the transition from G1 to S, thereby preventing cell proliferation. Our current knowledge of pRb is summarized in chapter 1.
Many chemotherapeutic drugs used in clinical settings target key
molecules regulating DNA synthesis or modulating the response to
DNA damage. Great progress has been made recently in characterizing the structure and functions of proteins responsible for activating
checkpoints that monitor DNA integrity, and many pharmaceutical
companies have explored molecular targets that control the S phase
checkpoint for therapeutic leads. Detailed discussions of the major
molecular players affecting DNA synthesis and the response to DNA
damage, from the viewpoint of biology and cancer drug development,
are provided in chapters 2 through 6.
Many checkpoints monitor the successful execution of the various
stages of mitosis to ensure that the cell produces two daughter cells with
identical genetic contents. Several protein kinase families are critical
for the regulation of these checkpoints. Since the 1990s, intensive drug
screening, coupled with studies of kinase structure and function, have
led to the identification of a variety of chemical compounds that are
capable of inhibiting individual mitotic kinases. Chapters 7 through 12
are devoted to advances in these areas.
Posttranslational modifications of the histone tails are closely
associated with regulation of the cell cycle as well as chromatin
structure. Multiple histone tail acetylations result in the destabilization
and decondensation of chromatin fibers. Given that histone acetylation
and deacetylation play an important role in the regulation of gene
expression and eventually the fate of the cell, much effort has been
made to develop histone deacetylase inhibitors to serve as anticancer
agents. Some of these inhibitors may affect the regulation of cell-cycle
checkpoints. Our current knowledge of histone deacetylase inhibitors
is summarized in chapter 13.
The overall goal of Checkpoint Responses in Cancer Therapy
is to enhance our understanding of the many cell-cycle checkpoint
molecules that have already been identified as effective targets
for anticancer drug development, and to explore the possibility of
developing a new generation of anticancer drugs with improved
therapeutic indices based on their ability to target a number of
checkpoint components that are yet to be fully studied. It is anticipated
that this book will serve as a valuable source of information, not only
for researchers in the pharmaceutical and biotechnology industries,
but also for academic scientists studying cell-cycle regulation, signal
transduction, and apoptosis, as well as those involved in cancer
Wei Dai
Tuxedo, NY
Preface .......................................................................................
Contributors ...............................................................................
1 RB-Pathway: Cell Cycle Control and Cancer Therapy.....
Erik S. Knudsen and Wesley A. Braden
2 Targeting the p53/MDM2 Pathway for Cancer Therapy ... 19
Christian Klein and Lyubomir T. Vassilev
3 DNA Topoisomerases as Targets for the
Chemotherapeutic Treatment of Cancer .......................... 57
Ryan P. Bender and Neil Osheroff
4 Targeting ATM/ATR in the DNA Damage Checkpoint.... 93
Joseph M. Ackermann and Wafik S. El-Deiry
5 Compounds that Abrogate the G2 Checkpoint................... 117
Takumi Kawabe
6 CDK Inhibitors as Anticancer Agents ................................ 135
Timothy A. Yap, L. Rhoda Molife,
and Johann S. de Bono
7 CHFR as a Potential Anticancer Target ............................. 163
Minoru Toyota, Lisa Kashima, and Takashi Tokino
8 Antimicrotubule Agents ...................................................... 177
Miguel A. Villalona-Calero, Larry Schaaf,
and Robert Turowski
9 Kinesin Motor Inhibitors as Effective
Anticancer Drugs.............................................................. 207
Vasiliki Sarli and Athanassios Giannis
10 Targeting the Spindle Checkpoint in Cancer
Chemotherapy................................................................... 227
Jungseog Kang and Hongtao Yu
11 Antiproliferation Inhibitors Targeting Aurora Kinases ...... 243
Kishore Shakalya and Daruka Mahadevan
12 Plks as Novel Targets for Cancer Drug Design ................. 271
Wei Dai, Yali Yang, and Ning Jiang
13 Do Histone Deacetylase Inhibitors Target Cell Cycle
Checkpoints that Monitor Heterochromatin Structure?... 291
Brian Gabrielli, Frankie Stevens,
and Heather Beamish
Index .......................................................................................... 311
Joseph M. Ackermann, PhD • Laboratory of Molecular Oncology
and Cell Cycle Regulation, University of Pennsylvania School of
Medicine, Philadelphia, PA
Heather Beamish, PhD • Lions Research Fellow, Cancer Biology
Program, Centre for Immunology and Cancer Research, University
of Queensland, Brisbane, Queensland, Australia
Ryan P. Bender, PhD • Department of Biochemistry, Vanderbilt
University School of Medicine, Nashville, TN
Wesley A. Braden, MD • Department of Cell and Cancer Biology,
University of Cincinnati, Cincinnati, OH
Wei Dai, PhD • Department of Environmental Medicine, New York
University School of Medicine, Tuxedo, NY
Johann S. de Bono, MD, FRCP, MSc, PhD • Senior Lecturer and
Consultant, Section of Medicine, Institute of Cancer Research, Royal
Marsden Hospital, Drug Development Unit, Sutton, Surrey, UK
Wafik S. El-Deiry, MD, PhD • Radiation Biology Program,
Abramson Comprehensive Cancer Center, Associate Director for
Physician-Scientist Training, Hematology-Oncology Division,
University of Pennsylvania School of Medicine, Philadelphia, PA
Brian Gabrielli, PhD • NHMRC Senior Research Fellow, Head,
Cell Cycle Group Diamantina Institute for Cancer Immunology
and Metabolic Medicine, University of Queensland, R Wing,
Princess Alexandra Hospital, Brisbane, Queensland, Australia
Athanassios Giannis, PhD, MD • Institute of Organic Chemistry,
Leipzig University, Leipzig, Germany
Ning Jiang, PhD • Department of Pathology, New York Medical
College, Valhalla, NY
Jungseog Kang, PhD • Department of Pharmacology, University of
Texas Southwestern Medical Center, Dallas, TX
Lisa Kashima, MS • Department of Molecular Biology, Cancer
Research Institute, Sapporo Medical University, Sapporo, Japan
Takumi Kawabe, MD, PhD • President and CEO, CanBas Co. Ltd.,
Makiya, Numazu, Japan
Christian Klein, PhD • Pharma Research, Roche Diagnostics,
Penzberg, Germany
Erik S. Knudsen, PhD • Department of Cell and Cancer Biology,
University of Cincinnati, Cincinnati, OH
Daruka Mahadevan, MD, PhD • Department of Medicine,
Hematology/Oncology, Arizona Cancer Center,
The University of Arizona College of Medicine, Tucson, AZ
L. Rhoda Molife, MRCP, MSc, MD • Section of Medicine, Institute
of Cancer Research, Royal Marsden Hospital, Drug Development
Unit, Sutton, Surrey, UK
Neil Osheroff, PhD • Professor of Biochemistry and Medicine,
John G. Coniglio Chair in Biochemistry, Department of
Biochemistry, Vanderbilt University School of Medicine,
Nashville, TN
Vasiliki Sarli, PhD • Institute for Organic Chemistry, University of
Leipzig, Leipzig, Germany
Larry Schaaf, PhD • Clinical Treatment Unit, The Ohio State
University, Comprehensive Cancer Center, Columbus, OH
Kishore Shakalya, BSc, MS • University of Arizona Cancer
Center, Tucson, AZ
Frankie Stevens, PhD • Cancer Biology Program, Centre for
Immunology and Cancer Research, University of Queensland,
Brisbane, Queensland, Australia
Takashi Tokino, PhD • Department of Molecular Biology, Cancer
Research Institute, Sapporo Medical University, Sapporo,
Minoru Toyota, MD, PhD • Department of Molecular Biology,
Cancer Research Institute, Sapporo Medical University, Sapporo,
Robert Turowski, MBA, RPh • Departments of Internal
Medicine and Pharmacology, The Ohio State University,
Columbus, OH
Lyubomir T. Vassilev, PhD • Discovery Oncology, Roche Research
Center, Hoffmann-La Roche Inc., Nutley, NJ
Miguel A. Villalona-Calero, MD, FACP • Associate Professor
of Internal Medicine and Pharmacology, Division of Hematology
and Oncology, The Ohio State University Medical Center,
Columbus, OH
Yali Yang, PhD • The Ohio State University Medical Center,
Department of Environmental Medicine, New York University
School of Medicine, Tuxedo, NY
Timothy A. Yap, BSc, MBBS, MRCP • Section of Medicine,
Institute of Cancer Research, Royal Marsden Hospital, Drug
Development Unit, Sutton, Surrey, UK
Hongtao Yu, PhD • Associate Professor, Department
of Pharmacology, University of Texas Southwestern Medical
Center, Dallas, TX
Cell Cycle Control and Cancer
Erik S. Knudsen
and Wesley A. Braden
Dysregulation of the RB Pathway
in cancer
Cell Cycle Control Through
the RB-Pathway
Influence of the RB-Pathway
Genotoxic Therapies
Impact of RB Pathway on
Influence of RB on
Antimicrotubule Agents
Targeted Therapeutics and the
Impact of RB Pathway
The retinoblastoma tumor suppressor (RB) is a negative regulator
of cellular proliferation that impacts multiple facets of cell cycle
control. To this end, RB function is commonly abrogated in tumorigenesis, thereby contributing to the uncontrolled proliferation of
From: Cancer Drug Discovery and Development
Checkpoint Responses in Cancer Therapy
Edited by: W. Dai © Humana Press, Totowa, NJ,
a part of Springer Science+Business Media
Knudsen and Braden
cancer cells. However, it is becoming increasingly clear that RB
is not only involved in the etiology and progression of cancer, but
also modifies the response to specific therapeutic modalities. Here
we discuss the role of RB in cell cycle control as it relates to the
response to specific classes of therapeutic agents.
Key Words: Tumor suppressor; retinoblastoma; cyclins;
chemotherapy; molecularly targeted therapeutics; cell cycle; E2F;
The retinoblastoma tumor suppressor protein, RB, is subject to
functional inactivation in a multitude of different tumor types. The Rb
gene was identified based on bialleleic inactivation in the pediatric
eye tumor, retinoblastoma (1–3). Subsequent analyses demonstrated
that loss of heterozygosity of the Rb gene or histochemical deficiency
of RB protein occurs in a wide fraction of human cancers and is
heterogeneous in nature, depending on the specific tumor type (4–10).
In addition to direct effects on Rb gene expression, the RB protein
is subject to post-translational modifications that have a significant
impact on its function. For example, viral oncoproteins (E1A, E7,
T-Ag) can bind to and directly inactivate RB. Such proteins function by
a combination of sequestration and targeting RB for degradation as key
facets of their transforming activity (11–13). Additionally, RB activity
is compromised as a consequence of CDK-mediated phosphorylation
(14–16). These phosphorylation events modify RB conformation and
disrupt virtually all of the biochemical activities associated with the
tumor suppressor (17). Therefore, it is perhaps not surprising that
deregulated phosphorylation of RB is a key factor in human cancer
(15,18). Specifically, deregulation of CDK-activity occurs through the
overexpression of cyclin D1 or loss of the CDK4/6-inhibitor, p16ink4a,
and is believed to trigger the aberrant phosphorylation and hence
inactivation of RB (14–16,19–21). Importantly, loss of RB function is
found in many tumor types but typically in a nonredundant manner,
such that deregulation of RB phosphorylation (e.g., p16ink4a loss), or
RB loss, are mutually exclusive events (19–22). Thus, these studies
have hastened the need to understand how RB functions, and correspondingly if defining RB status in a particular tumor can be used as
a determinant for efficacious therapeutic intervention.
Chapter 1 / RB-Pathway
RB is a critical regulator of the G1/S transition of the cell cycle,
which is responsive to CDK/cyclin activity. Conventionally, RB inhibits
S-phase entry by binding to the E2F family of transcription factors
and preventing transcription of genes required for DNA replication
and productive passage through mitosis (23–26). When hypophosphorylated, RB is bound to the transactivation domain of multiple E2F
proteins, eliciting transcriptional repression of E2F-regulated genes
(27–29). To achieve promoter repression, RB recruits corepressors
such as histone deacetylases (HDACs), histone methyltransferases,
members of the SWI/SNF complex, and polycomb group proteins
to E2F-regulated promoters (30–37). Upon mitogenic signaling, RB
becomes hyperphosphorylated as a consequence of CDK-mediated
phosphorylation, leading to release of E2F and transcription of
downstream target genes (18). Through this action, it is generally
believed that phosphorylation of RB by CDK/Cyclins is essential
for entry into S-phase. Coordination between CDK/Cyclin kinase
activity and E2F-transcription define the RB-pathway as a module
that is functionally inactivated in most cancer (14–17,19–22,38).
Antimitogenic signals, which can be broadly defined as those that
impede cellular proliferation, often are dependent on RB to inhibit
cell cycle progression (15,38). Such signals, are often targeted in
existing cancer therapy or are under investigation in the context of new
therapies (39,40). For example, DNA damage elicits cell cycle checkpoints that are dominant to the function of mitogenic signals (41), and
DNA damaging agents represent a mainstay of current cancer therapy
(42,43). Under conditions of such anti-mitogenic signals, RB phosphorylation is prevented, leading to subsequent reactivation of the protein
(15,18,38). Strikingly, the mechanisms through which RB can become
activated are quite varied, involving effects on both CDK/cyclin
activity and potential dephosphorylation of RB by phosphatase activities (18,44,45). Such signaling pathways have led to a concerted
analysis of how RB activation functions in discrete phases of the cell
cycle. Additionally, RB status as a determinant of the response to each
signaling pathway and impact on therapeutic signaling is currently
being resolved.
Knudsen and Braden
2.1. The RB Pathway in S-Phase Control
DNA replication is a very tightly regulated process. As cells exit
mitosis and enter G1, origin recognition complexes (ORCs) are bound
to sites of potential replication firing and recruit 2 proteins, cdc6 and
cdt1, independently (46–50). Mini-chromosome maintenance proteins
(MCM2-7), which are thought to function as the replicative helicases,
are recruited by cdc6 and cdt1 to form the prereplicative complex
(preRC) (48,51–53). As the cell progresses into S-phase, a multitude
of other proteins necessary for DNA replication are subsequently
recruited to the preRC, allowing for maturation of the replisome
complex that facilitates duplication of the genome (48,49,54).
The RB pathway is a critical molecular node that impinges upon the
DNA replication machinery. More clearly, many of the genes necessary
for DNA replication have been shown to be regulated by the E2F
transcription factor (55,56). As such, loss or activation of RB activity
significantly impacts RNA levels, protein levels and activity associated
with discrete DNA replication factors. Several studies have dissected
the impact of RB on replication control, and these studies have
uniformly demonstrated that RB has the capacity to actively inhibit
DNA replication (57–59). Functional analyses of the mechanism underlying this action have illustrated that RB has the capacity to disrupt
PCNA chromatin association through a pathway associated with the
down-regulation of CDK2 activity. Consistent with this finding, several
laboratories have subsequently demonstrated that inhibition of CDK2
activity via distinct mechanisms, mediates similar influences on PCNA
function (57,59,60). More recently, it was determined that p16ink4a,
which signals upstream of RB to inhibit CDK4 activity, can inhibit
MCM chromatin association in G1 (58). Perhaps even more surprising
was that p16ink4a expression was able to inhibit total protein levels
of cdc6 and cdt1, whereas RB was not (58). Although RB has no
effect on replication control in G1, p16ink4a has no effect on replication in S-phase. This is most likely because of low CDK4 activity
in S-phase, thus compromising p16ink4a signaling. These data suggest
a fundamental difference by which RB and p16ink4a influence DNA
replication and underscore that although these proteins exist within the
same pathway, they have differential effects on cell cycle and replication control (61). In addition to these specific models of RB-mediated
arrest, RB controls the expression of a wide spectrum of replication
factors (e.g., MCM2, MCM5, cdc6, and PCNA) and thus, deficiency
of RB has the capacity to dramatically influence the coordination of
S-phase (57,62,63).
Chapter 1 / RB-Pathway
2.2. Involvement of RB Pathway in G2/M Control
Akin to the processes involved in DNA replication, progression
through mitosis is achieved via a number of discrete steps associated
with mitotic entry and subsequent exit from mitosis into G1. Mitotic
entry is dependent on the activation of CDK1/cyclin B complexes that
initiate nuclear envelope breakdown and chromosome condensation
(64,65). However, mitotic progression from metaphase is dependent
on passage through the spindle checkpoint. This checkpoint stalls
progression to anaphase, and ensures that all chromosomes are
associated with the mitotic spindle (66–68). Once all chromosomes
are bound by the spindle, specific signals coalesce to promote the
activation of the anaphase promoting complex (APC) to mediate
the destruction of multiple substrates and allow progression through
anaphase, and ultimately exit from mitosis (69).
The extent to which the RB-pathway contributes to G2/M progression
is less clearly defined than is the role of RB in other phases of the cell cycle.
Activation of RB by multiple means has not demonstrated a pronounced
effect on the progression through mitosis, as cells fail to accumulate in G2
or any mitotic stages (70). However, as is the case with DNA replication,
multiple critical factors that are required both for mitotic entry (e.g., Cdk1
and Cyclin B1) and exit (e.g., Cdc20, Plk1, and Mad2) are regulated by
RB (56,71–73). Thus, one would predict that the RB pathway impinges
on the regulation of mitosis. Consistent with this supposition, loss
of RB has recently been associated with delayed mitotic progression
occurring as a consequence of MAD2 upregulation (71).
The majority of cytotoxic regimens utilized in the treatment of
cancer cause DNA damage (74). For example, cisplatin, cyclophosphamide and ionizing radiation directly damage DNA, whereas agents
such as the topoisomerase poisons, irinotecan, and doxorubicin induce
DNA damage as a result of indirect effects (75–79). Following such
treatment, S-phase is slowed or halted to allow for adequate repair of
the lesions before progressing into mitosis (80–83). Combined, these
controls limit the segregation of damaged DNA to daughter cells.
It has been shown by multiple groups that RB is required for the
solicitation of cell cycle arrest in the presence of DNA damage (17,45,
84,85). Initially, it was observed that RB-deficient embryonic fibroblasts failed to undergo the cessation of DNA replication following
Knudsen and Braden
DNA damage (85,86). However, these cells still mount a G2/M checkpoint response, and as such fail to progress through mitosis and harbor
a propensity to over-replicate their genomes (45,87). Using RNAi
technology, RB was subsequently shown to play similar roles in
response to cytotoxic therapy in tumor cells, whereas other components of the RB pathway (e.g., p16ink4a loss) did not influence the
checkpoint response (84). The consequence of RB loss in both fibroblasts and tumor cell models is associated with increased sensitivity
to DNA damaging agents (84,85,88). Thus, although RB loss enables
checkpoint bypass, invariably this has been associated with enhanced
cell killing. The mechanisms through which RB influences survival are
not intrinsically clear, but could be dependent on additional secondary
damage as a consequence of ongoing DNA replication in the presence
of damage (89). Alternatively, it has been reported that RB loss deregulates the expression of many proapoptotic factors, thus shifting the
balance toward cell death following DNA damage (90–92). Irrespective
of the mechanism, these findings suggest that RB-deficient tumors may
be particularly amenable to treatment with cytotoxic agents. However,
an ongoing concern would be that tumors lacking checkpoint function
would be genomically unstable, and thus although initial therapy may
be quite effective, mutations afforded at the time of treatment could
contribute to rendering the tumor more aggressive and ultimately
therapy resistant.
Some of the most commonly utilized agents in the treatment
of cancer are anti-metabolites that largely function by perturbing
dNTP pools (93,94). For example, the thymidylate synthase inhibitors
(5-fluorouracil, tomudex) are first line agents in the treatment of
many cancers and have a pronounced deleterious effect on DNA
replication (93). Interestingly, RB controls many facets of DNA
replication, including the levels of many metabolic enzymes, such
as thymidylate synthase, dihydrofolate reductase and ribonucleotide
reductase subunits (60,95–99). As such, RB-deficient cells express
highly elevated levels of these enzymes and RB has the capacity to
dramatically alter dNTP pools (97). In cell culture models, RB loss
enables ongoing DNA replication in the presence of such agents,
suggesting that RB-deficiency negatively affects therapeutic response
Chapter 1 / RB-Pathway
A number of chemotherapeutic agents function by perturbing
microtubule dynamics and correspondingly the function of the
mitotic spindle. Specifically, taxanes and vinca-alkaloid agent are
utilized clinically and prevent appropriate progression through mitosis
(100–104). A consequence of this protracted mitotic-deficit is cell
death, making these compounds very effective cytotoxic agents. As
discussed above, there is evidence that RB-status impacts the regulation
of mitotic progression. Taxanes promote microtubule stability and have
been shown to influence RB phosphorylation (105–107). However,
vinca-alkaloids, which destabilize microtubules, are one class of
compounds for which the absence of RB has little effect on cell
cycle response (86). In contrast, RB deficiency facilitates ongoing
DNA replication and changes in cell ploidy following treatment with
nocodazole that also destabilizes microtubule arrays (108,109). Strikingly, there is little indication of how RB loss ultimately modifies
sensitivity to these clinically relevant agents.
Recently, there has been a dramatic initiative to define anti-cancer
agents that function upon specific molecular targets. Such therapeutics
currently encompass a wide-range of targets (e.g., steroid hormone
receptors, growth factor signaling pathways, cell cycle machinery, and
chromatin). Many of these agents function by antagonizing mitogenic
signaling cascades and thus inhibiting proliferation of tumor cells. As
such, it may be suspected that loss of RB would have a significant
impact on the response elicited downstream of such agents. Here we
will briefly discuss several targeted therapeutics for which the role of
RB has been investigated:
6.1. Staurosporine and 7-Hydoxystaurosporine
Staurosporine and related analogs where classically believed to
function as general PKC inhibitors. However, such agents impact
a diverse range of targets including cyclin dependent kinases and
Chk1 (110). Studies have demonstrated that cells containing functional
RB respond to staurosporine treatment, resulting in diminished CDK4
activity and G1 arrest (110). Additionally, RB is required for
Knudsen and Braden
staurosporine to elicit this G1 arrest (111). Similar data exists for
7-hydroxystaurosporine (UCN-01) implying RB-status is a key determinant of cell cycle response (112). In the case of UCN-01, RBdeficiency and corresponding cell cycle progression is associated with
enhanced cell death (113).
7-Hydroxystaurosporine (UCN-01), which was originally identified
as a protein kinase C selective inhibitor, is currently in clinical trials as
an anticancer drug (114). It has been previously shown that UCN-01
induced preferential G1-phase accumulation in tumor cells and this
effect was associated with the RB and its regulatory factors, such as
CDK2 and CDK inhibitors p21Cip1 and p27Kip1 (115). However,
recent evidence suggests UCN-01 arrests cells in G1 regardless of
RB-status (113). Perhaps more striking was that UCN-01 induced
apoptosis in RB-deficient cell lines, but not in RB-proficient cell
lines (113). These observations suggest that G1-checkpoint function
might be important for cell survival during UCN-01 treatment (116).
Combined, the data implicate a functional RB pathway as a significant
determinant of the sensitivity of tumor cells to UCN-01.
6.2. Geldanamycins
Geldanamycin (GM) is an ansamycin antibiotic that functions to
inhibit heat shock protein 90 (Hsp90) chaperone function and results
in the subsequent degradation of important signaling molecules (e.g.,
cyclin D1). Although GM has been shown to diminish CDK2 and
CDK4 activity resulting in G1 arrest in RB proficient backgrounds,
GM had no effect on cell cycle kinetics in an RB negative background
(117). Furthermore, release of RB negative cells from G2/M in the
presence of GM continued through G1 and the subsequent cell cycle
unperturbed (117). Thus, RB is necessary for effective GM treatment
of human tumors.
6.3. CDK Inhibitors
Flavopiridol, roscovitine, PD0332991 and other compounds in
development inhibit CDK activity, preventing phosphorylation of
various CDK target proteins and ultimately leading to cell cycle arrest
(118). Although flavopiridol functions as a generic CDK inhibitor
molecule, roscovitine can inhibit CDK1 and CDK2 function, and
PD0332991 specifically inhibits CDK4 kinase activity. In xenograft
models, these agents halt tumor growth and can reduce tumor volume
(114,118,119). Although these results are promising, RB status has
Chapter 1 / RB-Pathway
been shown to play a critical role in response to these agents. Particularly, the growth suppressive functions of PD0332991 are entirely
dependent on RB, such that RB-deficient tumor models fail to respond
to treatment. Therefore, understanding the functional status of RB
will be an absolute requirement for the effective usage of such
agents (58).
Combined, these studies indicate that RB status is a determinant of a
variety of therapeutic approaches utilized in the treatment of cancer. In
certain instances, RB-deficiency is associated with increased sensitivity
to specific agents. This is most clearly established in the case of specific
genotoxic agents. However, in other instances, RB-deficiency has an
apparent deleterious impact on response, as exemplified with therapies
directed at CDK activity. These studies provide an important basis
for further investigation of RB action on therapeutic response and for
devising methodologies to most efficaciously treat cancer rationally.
However, to build upon these basic findings will require a number of
further advances that will facilitate the utilization of RB-status as a
determinant of therapeutic response.
The authors regret any omissions in the preparation of this article
and thank our colleagues for though-provoking discussion. Members
of Karen Knudsen’s and Erik Knudsen’s laboratories assisted in the
preparation and editing of the manuscript.
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Targeting the p53/MDM2
Pathway for Cancer
Christian Klein
and Lyubomir T. Vassilev
The p53 Tumor Suppressor Protein
Reactivation of Mutant p53 in
Inhibition of the p53-MDM2
Inhibition of MDM2 E3 Ligase
Modulation of p53 Activity for
Protection of Normal Tissues
During Chemotherapy
Key Words: 53; MDM2; tumor suppressor; genome guadian;
protein-protein interaction
Nearly 40,000 research and review articles published since 1980
have elevated the tumor suppressor p53 to the rank of the most studied
protein in cancer research (1–5). However, the translation of this
From: Cancer Drug Discovery and Development
Checkpoint Responses in Cancer Therapy
Edited by: W. Dai © Humana Press, Totowa, NJ,
a part of Springer Science+Business Media