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Jennifer A. Pietenpol
Michael B. Kastan
CONTROL OF THE CELL
5
CYCLE
SUMMARY OF KEY POINTS
" Cells in most postnatal tissues are occurs in late G1 and is the point " Arrests in G2 and M phases
quiescent. Exceptions include cells of beyond which the cell is committed after DNA damage or mitotic
the hematopoietic system, skin, and to progress through the rest of the spindle damage are presumably to
gastrointestinal mucosa. cell cycle. prevent propagation of damaged
" Two major challenges for " Cell cycle checkpoints are chromosomes and to ensure
proliferating cells are to make an surveillance mechanisms that link appropriate segregation of
accurate copy of the three billion the rate of cell cycle transitions to chromosomes to daughter cells.
bases of DNA (S-phase) and to the timely and accurate completion " Disruption of cell cycle controls is a
segregate the duplicated of prior, dependent events. pathognomonic feature of all
chromosomes equally into daughter " Cells can arrest at cell cycle malignant cells. Disruption can
cells (mitosis). checkpoints temporarily to allow manifest as alterations of growth
" Progression through the cell cycle is for (a) the repair of cellular factor signaling pathways,
dependent on both extrinsic and damage; (b) the dissipation of an dysregulation of cyclin protein
intrinsic factors. exogenous cellular stress signal; or expression, enhanced activity of
" Extrinsic factors include cell-to- (c) availability of essential growth cyclin-dependent kinases, altered
cell contact, basement membrane factors, hormones, or nutrients. expression or function of cyclin-
attachments, and growth factor or " Among the major functions of the dependent kinase inhibitors, and
cytokine exposure. p53 tumor suppressor protein are mutation of cell cycle checkpoint
" The internal cell cycle machinery to modulate cellular responses to controls.
is controlled largely by oscillating stress and to induce cell cycle " Because cell cycle control is
levels of cyclin proteins and by arrest, senescence, or death as disrupted in virtually all tumor
modulation of cyclin-dependent appropriate. types, the cell cycle-related gene
kinase activity. " A major objective of cell cycle products that are mutated in tumors
" One way in which growth factors arrests in the G1 and S phases of provide therapeutic targets that
regulate cell cycle progression is by the cell cycle after DNA damage might preferentially affect tumor
affecting the levels of cyclin D in the is presumably to minimize cells more than normal tissues.
G1 phase of the cell cycle.The replication of damaged DNA
 restriction point of the cell cycle templates.
conserved signal transduction system for controlling cell
INTRODUCTION cycle transitions through regulation of the activity of key
enzymes called cyclin-dependent kinases. Further, many
Although most cells in an adult human are quiescent or in investigations have focused on understanding how the
a nonproliferative state, specialized cells, such as those of signaling pathways that mediate the cell cycle transitions
the hematopoietic system or those that line the gastro- are regulated and modified after cellular stresses. Human
intestinal tract, maintain proliferation. On average, about cells are continuously exposed to external agents (e.g.,
two trillion cell divisions occur in an adult human every reactive chemicals and UV light) and to internal agents
24 hours (about 25 million per second). It is critically (e.g., byproducts of normal intracellular metabolism, such
important that various cell types divide at a rate sufficient as reactive oxygen intermediates) that can induce cell
to produce the needed cells for growth and replacement. stress. Eukaryotic cells have evolved cell cycle machinery
If, however, any given cell type divides more rapidly than with a series of surveillance pathways termed cell cycle
is necessary, the normal organization and functions of the checkpoints to ensure that cells copy and divide their
organism will be disrupted, as the rapidly dividing cells genomes with high fidelity during each replication cycle.
invade and interfere with specialized tissues. Such is the Cell cycle arrest after DNA damage is critical for
course of events in cancer. maintenance of genomic integrity, and loss of normal cell
Over the past two decades, unraveling the basic cycle checkpoint signaling is a hallmark of tumor cells.
molecular events that control eukaryotic cell cycle transi- The ability to manipulate cell cycle checkpoint signaling
tions has been an area of intense research pursuit. Studies also has important clinical implications, as modulation of
in a variety of organisms have identified an evolutionarily the checkpoints in human tumor cells could enhance
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cellular sensitivity to chemotherapeutic regimens that cell s macromolecules and organelles are also duplicated
induce DNA damage. This chapter focuses on the and partitioned, more or less evenly, between daughter
mechanics of the cell cycle and checkpoint signaling cells. During normal cell proliferation, these two cycles
pathways and on how this knowledge might lead to more occur at the same rate, so that each round of DNA
efficient use of current anticancer therapies and to the synthesis and mitosis is balanced by doubling of all other
development of novel agents. macromolecules in the cell. In this way, the DNA/protein
ratio of a cell is maintained within advantageous limits.
Orderly progression through the cell cycle is ensured by
THE CELL CYCLE MACHINERY intrinsic mechanisms that regulate the dependence of one
cell cycle event on another. For example, replication of
DNA cannot take place until cells have passed through
Overview of Cell Cycle Phases
mitosis. In addition, regulatory controls, called check-
The cell cycle is the sequence of events by which a points, can modulate cell cycle progression in response to
growing cell duplicates all its components and divides adverse conditions (such as those in the presence of
into two daughter cells, each with sufficient machinery to damaged DNA) and will be discussed in a subsequent
repeat the process. The most important components are section. When cells encounter specific growth inhibitory
the cell s chromosomes, which contain DNA in complex signals or there is an absence of appropriate mitogenic
with proteins.Eukaryotic cell division is a highly regulated signaling, cells can cease proliferation and enter a
process. One round of cell division requires high-fidelity nondividing, quiescent state known as G0, or they can
duplication of the three billion bases of DNA in each undergo apoptosis.
cell during S phase of the cell cycle and proper
segregation of duplicated chromosomes during mitosis or
Mechanics of the Cell Cycle Engine
M phase. Before and after S phase and M phase the cell
transits through  gap phases, termed G1 and G2 (Fig. 5-1). Cell cycle progression is mediated by the activation of a
G1 phase is a period after mitosis when cells prepare for highly conserved family of protein kinases, the cyclin-
successful DNA synthesis, and G2 is a period after DNA dependent kinases (cdks).1,2 Activation of a cdk requires
synthesis when the cell prepares for successful mitosis. binding to a specific regulatory subunit, termed a cyclin.
The cycle of DNA synthesis and sister chromatid separa- Cyclins were so named because of their fluctuating levels
tion runs in parallel with a growth cycle in which the through the cell cycle.The presence of a 100-amino acid
sequence, the  cyclin box, defines a protein as a cyclin
family member.1 The cyclin/cdk complexes are the central
cell cycle regulators, with each complex controlling a
specific cell cycle transition (see Fig. 5-1).To date, at least
New daughter cell
nine cdks and 15 cyclins have been described.3 Extra-
cellular stimuli, such as growth factors and hormones,
elevate D-type cyclins (cyclins D1, D2, and D3), which
bind to and activate cdk4 and cdk6 and stimulate
Mitosis (cell division)
Begin cycle
quiescent cells to enter the cell cycle or proliferating
cells to continue proliferation.4 7 After elevation of D-type
Cyclin B/cdc2
M
cyclins and activation of cdk4 or cdk6 in G1, cyclin E
levels increase and bind cdk2 in cell. The cyclin E/cdk
G2
complexes regulate the transition from G1 into S phase.
Cyclin A/cdc2
G1 Cyclin D/cdk4,6 8 10
Cyclin A is induced shortly after cyclin E and binds to
cdk2 in S phase and to cdc2 (cdk1) in G2 and mitosis.11
Cyclin A is thought to be involved in the regulation of S
DNA synthesis
S
phase entry, and it is also important in G2 and M phases.12
(doubling of DNA)
The entry into mitosis from G2 is under the control of
Cyclin A/cdk2
B-type cyclins, which also associate with cdc2.13 15
Cyclin E/cdk2
In normal cells, the cdks are expressed throughout
Restriction point
the cycle; however, each cyclin protein has a restricted
period of expression. The limited expression of each
Figure 5-1. One round of cell division requires high-fidelity duplication cyclin protein is due to cell cycle-dependent regulation of
of DNA during S phase of the cell cycle and proper segregation of
both cyclin gene transcription and protein degrada-
duplicated chromosomes during mitosis or M phase. Before and after S
tion.16,17 For cdks to become active, they must bind a
phase and M phase, the cell transits through  gap phases, termed G1
cyclin and undergo site-specific phosphorylation. The
and G2. Extracellular stimuli, such as growth factors and hormones,
cyclin/cdk complex is regulated by a number of
elevate D-type cyclins that bind to and activate cdk4 and cdk6 and
stimulate cells to transit through G1 to the restriction point.At the
phosphorylation and dephosphorylation events, resulting
restriction point, cyclin E levels increase and bind cdk2 in the cell.The
either in activation or inhibition of kinase activity.1
cyclin E/cdk complexes regulate the G1/S transition. Cyclin A is induced
Phosphorylation is carried out by cyclin-activating kinase,
after cyclin E and binds cdk2 in S phase and cdc2 in G2 and mitosis.
and dephosphorylation is mediated by members of the
The entry into mitosis is under the control of B-type cyclins, which also
associate with cdc2. Cdc25 family of dual-specificity protein phosphatases.
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5 " Control of the Cell Cycle 3
The mammalian Cdc25 family consists of three members:
Cdc25A, Cdc25B, and Cdc25C, which appear to have
cyclin D cyclin D
specificity for different cyclin/cdk complexes.18 20
CAK P
cdk4,6
P P
Cdc25A promotes entry into S phase by acting on cyclin
cdk4,6
A/cdk2 and cyclin E/cdk2 and is required for DNA P
P
RB
P P
replication.21 24 Further, Cdc25A is a transcriptional target
P
P
of c-Myc and E2F,and its RNA and protein levels increase as
RB
cells are stimulated to enter the cycle from quiescence.24 26
RB RB
Another important regulator of S phase progression is the
Cdc25B phosphatase.27 Cdc25B activation occurs during
E2F E2F E2F
S phase and peaks during the G2 phase, and Cdc25B
activity is necessary for S phase completion in vivo.28,29 S, G2,
and M
Both Cdc25B and Cdc25B play roles in the G2/M
phase
transition. Cdc25C dephosphorylates cyclin B1/cdc2 and
cyclin E cyclin E E2F genes
is essential for progression through the G2/M phase of the
CAK
cdk2
cell cycle.18,30 Cdc25B appears to play a similar role, but Cyclin E
cdk2
Cyclin A
with a different timing with respect to Cdc25C.31
P
Cyclin B
DHFR
G1 Phase
TK
G1 is a phase in which cells make critical decisions about
their fates, including the commitment to replicate DNA
Figure 5-2. The G1/S transition. During G1 phase progression, activation
and complete the cell division cycle. If mitogens are avail-
of cyclin D/cdk4 and cyclin E/cdk2 complexes by cyclin activating
able and the cellular milieu is favorable for proliferation, a
kinase (CAK) leads to sequential phosphorylation of the transcription
decision to enter S phase is made at a time in mid-to-late factor RB. Hypophosphorylated RB binds to the E2F transcription factor
family to inhibit S phase entry. Once hyperphosphorylated, RB
G1, called the restriction point (see Fig. 5-1). In unstressed
dissociates from E2F, resulting in activation of genes required for S
cells, this commitment to replicate DNA and divide is
phase entry.
irreversible until the next G1 phase.The restriction point
switch, from the growth factor-dependent early G1 to the
subsequent mitogen-independent phases, reflects the
induction of broad transcription programs that regulate ylation is necessary for the successful completion of
genes critical for G1/S transition and coordination of S-G2- DNA replication.41 Cell cycle regulation by RB plays an
M phase progress. important role in preventing tumorigenesis, as mutations
Integral to the molecular switch that controls transition that affect the RB signaling pathway have been identified
from G1 to S phase and key downstream targets of the G1 in the majority of human cancers.42
phase cyclin/cdk complexes are the members of the In addition to regulation by phosphorylation and
retinoblastoma protein (RB) family: RB, p107, and p130.32 dephosphorylation events as described previously, cdks
During G1 progression, RB is sequentially phosphorylated are regulated by a group of functionally related proteins
by cyclin D1/cdk4,6 and cyclin E/cdk2 complexes called cdk inhibitors.1 The cdk inhibitors fall into two
(Fig. 5-2).33 35 Phosphorylated RB can function as either families: the INK4 inhibitors and the Cip/Kip inhibitors.
a transcriptional repressor or a transcriptional activator There are four known INK4 family members: p16INK4A,
depending on its phosphorylation state and the proteins p15INK4B, p19INK4D, and p18INK4C, and three known Cip/Kip
with which it binds.36 Best understood is the role of RB family members:p21Waf1/Cip1,p27Kip1,and p57Kip2.The INK4
as a transcriptional repressor in its hypophosphorylated family specifically inhibits cdk4 and cdk6 activity during
state when bound to the E2F family of transcription the G1 phase of the cell cycle, while the Cip/Kip family
factors.36 The E2F family mediates transcription of genes can inhibit cdk activity during all phases of the cell cycle
required for DNA synthesis, including cyclin E, cyclin A, (Fig. 5-3). Both families of cdk inhibitors can arrest cells in
cyclin B, dihydrofolate reductase, and thymidine kinase the G1 phase of the cell cycle by inhibiting the activities
(see Fig. 5-2).37 The binding of hypophosphorylated RB to of cdks and preventing their ability to phosphorylate and
E2F inhibits E2F-dependent transcription of S phase genes inactive RB and other RB-family proteins.1 The levels,
and arrests cells at the G1/S transition.38 The ability of subcellular localization,and activity of these inhibitors can
RB to function as a transcriptional repressor involves be regulated by various forms of cell stress and growth
other protein families, including histone deacetylase and inhibitory signaling pathways.
chromatin remodeling SWI/SNF complexes.39 RB can also
be regulated by acetylation, which is mediated by histone S Phase
acetylases such as p300/CBP. The acetylases are under cell Biochemical and genetic approaches have brought major
cycle control and prevent efficient RB phosphorylation by advances to our understanding of how DNA replication
cyclin E/ckd2.40 Sequential phosphorylation of RB by is controlled in the cell. The quest for the molecular
cyclin D/cdk4/6 and cyclinE/cdk2 complexes inhibits the mechanisms that ensure genome integrity by controlling
repressor activity of RB, as it results in the dissociation of once-per-cell cycle replication has resulted in the emer-
E2F and RB, and S phase entry (see Fig. 5-2). As cells gence of a fundamental model describing the control of
progress into S phase, maintenance of RB hyperphosphor- DNA synthesis. Eukaryotic DNA replication is a complex
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Cdt1 are required to load MCM proteins on chromatin.50,51
Human Cdt1 is coassociated with geminin, which is a
cyclin D1 cyclin D1
negative regulator of pre-RC formation that prevents the
Cdk4
loading of MCM onto chromatin.50 This sequential asso-
cdk4,6
P
p16
P P P
ciation of initiator proteins with origin DNA licenses
p27
chromatin for replication.
P
pRB
P P
A model has been proposed to explain the coordination
p21
P
of chromatin licensing and the cell cycle (see Fig. 5-4).The
P
pRB
ORC associates with replication origins, and this associa-
pRB pRB
tion persists throughout the cell cycle.52 As cells complete
mitosis, Cdc6 and Cdt1 are loaded on chromatin, and they
E2F E2F E2F
in turn load the MCM complex on chromatin, at which
point licensing is considered complete.50The multiprotein
p21
complex is considered to be the pre-RC.43 This complex
is activated at the G1/S transition, and DNA replication
p27
is initiated. Two protein kinases, cdk2 and HsDbf4-
E2F
dependent kinase (hcdc7), are required to activate the
cyclin E cyclin E
licensed origins for initiation. The activity of the protein
Cdk2
kinases is believed to result in changes in the pre-RC that
Cdk2
promotes Cdc45 binding to the MCM complex, followed
P
by the unwinding of replication origins. Subsequently,
DNA replicating proteins such as RPA, DNA polymerase Ä…
Figure 5-3. Role of cdk inhibitors in the G1/S transition. Members of
and µ are recruited to initiation sites.53 55 After activation of
the INK4 and Cip/Kip cdk inhibitor families (represented by p16 and
p21/27, respectively) can inhibit the cyclin/cdk kinase complexes to the replication origins,both the MCM complex and Cdc45
mediate a G1/S cell cycle arrest. INK4 family members bind directly to
move together with replication enzymes assembled at
cdk4 or cdk6 and inhibit binding to the cyclin, whereas p21 and p27
replication forks to complete DNA replication.56,57 Thus,
can bind to both the cyclin and cdk components with higher affinity
an increase in cdk2 and hcdc7 activity at the G1/S
for the cyclin.
transition triggers initiation and converts the origin to the
post-RC state. The reformation of pre-RC does not occur
process including the recognition of initiation sites on again until the end of mitosis.
DNA, multistep preparation of DNA for duplication, and To maintain genomic integrity, it is essential that origins
assembly of multiprotein complexes capable of beginning do not fire a second time until mitosis has been com-
DNA synthesis at initiation sites. The process starts late pleted.The cdk cycle controls the two states at replication
in M phase as the cell completes its previous cycle and origins,couples the initiation of S phase to the completion
lasts until the appropriate time of DNA replication at of M phase, and prevents re-replication events from
each origin-of-replication initiation site. An interplay of occurring during a single round of the cell cycle.58 60 The
multisubunit protein complexes occurs during S phase, MCM complex is a key component of DNA replication;the
involving proteins that bind to the origins of replication cycle of cdk activity within the cell regulates the precise
initiation, proteins with helicase activity, replication timing of loading and activation of the MCM complex
protein A, and DNA polymerase Ä…. and prevents its reloading before the completion of a cell
The initiation of DNA replication during the S phase cycle.
of the cell cycle takes place at multiple sites on the
chromosomes, called the origins of replication.The state G2/M Phase Transition
of eukaryotic replication origins changes during the cell After duplication of the genome in S phase, cells transit
cycle (Fig. 5-4).43,44 It is proposed that replication origins through G2 and prepare for mitosis. As cells enter into
are in two different states during the cell cycle. One state G2 phase, cyclin B/cdc2 complexes form and are kept
exists during G1 phase, before DNA replication begins, inactive by phosphorylation (Fig. 5-5). At the end of G2
when a multiprotein complex called the prereplicative phase, cyclin B/cdc2 complexes are activated by dephos-
complex (pre-RC) assembles on the origin. The second phorylation, and cells enter into mitosis.61,62 Phosphoryla-
state exists from the initiation of S phase to the end of M tion is carried out by the Wee1/Mik1 family of protein
phase,when a postreplicative complex (post-RC) is present kinases.63,64 The enzyme that dephosphorylates and
at the replication origins.Cdk activity is thought to control activates cdc2 at the end of G2 and initiates mitosis is
65,66
each round of DNA replication.At the end of M phase, low Cdc25C. Cdc25C is localized in the cytoplasm during
cdk activity allows for the assembly of the pre-RC, a state interphase and enters the nucleus just before mitosis.19
competent for replication. When chromatin becomes Although less well understood, Cdc25B also plays a role at
replication-competent,it is referred to as licensed.Initiator the mitotic transition.The cyclin A/Cdc2 complex is likely
proteins required for pre-RC formation include the Origin regulated in a similar manner; however, further studies are
Recognition Complex (ORC), Cdc6, Cdt1, and MCM necessary to define the role of cyclin A kinase activity in
proteins (MCM2 to MCM7).45 48. The six MCM proteins mitosis.
interact with each other to form a hexameric complex Another mechanism by which cyclin B1/cdc2 com-
thought to function as a replicative helicase.49 Cdc6 and plexes are regulated is through a shift in their subcellular
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5 " Control of the Cell Cycle 5
Figure 5-4. S phase transition.
Geminin
The origin replication complex
degradation
(ORC) associates with replication
origins, and this association
Dephosphorylation
persists throughout the cell
cycle.As the cells complete
P
mitosis, Cdc6 and Cdt1 are loaded
on chromatin, and they in turn
load the MCM complex on
Cdt1
cdc6
MCM2-7
chromatin, at which point
P
licensing is considered complete ORC
ORC
and the multiprotein complex is pre-RC
considered to be the pre-RC.This
P
post-RC
complex is activated at the G1/S
cdc6
transition, and DNA replication is
initiated.Two protein kinases
P
cdk and HsDbf4-dependent
MCM2-7
kinase are required to activate
Licensed state
the licensed origins for initiation.
P
cdc45
The activity of the protein P
Replication
kinases is believed to result in
machinery
changes in the pre-RC, which
promotes Cdc45 binding to the P
ORC
MCM complex, followed by the
cdk
P
unwinding of replication origins. cdc6 hcdc7
Geminin
Cdt1
Subsequently, DNA replicating
Degradation
P
proteins such as RPA, DNA
Cdt1
or export
Degradation
polymerase Ä…, and DNA
or export
polymerase µ are recruited to
initiation sites.An increase in cdk
and hsDbf4 activity at the G1/S
transition triggers initiation and
converts the origin to the post-
RC state.The reformation of pre-
RC does not occur again until the
end of mitosis.
localization. For most cyclins, the scenario after biosynthe- 5. During telophase, the parent cell is divided into two
sis is nuclear localization until cell cycle-mediated degra- daughter cells by cytokinesis.
dation occurs. In contrast, cyclin B1 is in the cytoplasm
As cells enter mitosis, phosphorylation of key compo-
during S phase and G2 phase and is translocated to the
nents causes significant changes in the architecture of the
nucleus at the beginning of mitosis.67 It is thought that
cell.This phosphorylation is due mainly to cyclin B/cdc2
the precise regulation of cyclin B1 localization prevents
activity.68 Cyclin B/cdc2-mediated phosphorylation
premature mitosis during interphase, while allowing
induces changes in the microtubule network, the actin
regulated access of cyclin B1/cdc2 complexes to their
microfilaments, and the nuclear lamina.69 71 Other cyclin
nuclear substrates at the onset of mitosis.
B/cdc2 substrates include histone H1 and microtubule-
associated proteins such as MAP4, MAP2, and
M Phase
stathmin.16,72 In addition to the central function of cdc2,
Mitosis is the process by which a cell ensures that each
the family of polo-like protein kinases (Plks) also plays
daughter cell will have a complete set of chromosomes.
a critical role in several mitotic events.73,74 Several Plk
There are five key stages of mitosis (Fig. 5-6):
homologs have been identified in mammalian cells.75
1. During prophase, the chromosomes become con- In human cancer cells, injection of anti-Plk1 antibodies
densed, and proteins begin to bind the kinetochores, leads to a mitotic arrest with a monopolar spindle formed
preparing for spindle attachment. around a smaller than usual centrosome.76 These findings
2. Upon breakdown of the nuclear envelope, the cell suggest that Plks are critical for the formation of a bipolar
enters prometaphase, during which the mitotic spindle spindle. It is proposed that Plks initiate the onset of
is formed and the chromosomes attach to microtubules mitosis by activating Cdc25C, although little is known
in the spindle through their kinetochores. Once about the exact trigger for Plk activation.77 Plks are also
attached, the chromosomes align along the metaphase important regulators of mitotic exit.
plate in the center of the spindle. Mitotic exit requires sister chromatid separation,
3. During metaphase,all of the chromosomes are attached spindle disassembly, and cytokinesis. The initiation and
to microtubules through their kinetochores and are coordination of these processes are controlled by degra-
aligned at the metaphase plate. dation of key regulatory proteins. The mediator of this
4. At anaphase onset, the sister chromatids separate and protein destruction is a multisubunit protein called the
move toward the poles of the spindle. anaphase-promoting complex (APC) or cyclosome.78,79
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growth factor availability and cell mass accumulation also
;
regulate cell cycle transition.86 Cells can arrest at cell cycle
G2 P P P Cdc25C M checkpoints temporarily to allow for any of the following:
P
" The repair of cellular damage
Inactive cdc2 cdc2 Active
Spindle assembly " The dissipation of an exogenous cellular stress signal
cyclin B cyclin B
Chromatin condensation
" The availability of essential growth factors, hormones,
Nuclear envelope
or nutrients
breakdown
Wee1 Tyrosine-15
The best studied of the cell cycle checkpoints are those
Cyclin
cyclin B
that monitor the status and structure of chromosomal
Myt1 Threonine-14
degradation
DNA during cell-cycle progression. These checkpoints
CAK Threonine-161
contain, as their most proximal signaling elements, sensor
proteins that scan chromatin for partially replicated DNA,
Inactive cdc2 cdc2 Inactive
DNA strand breaks,or other abnormalities.Sensor proteins
cyclin B are thought to translate DNA-derived stimuli into bio-
chemical signals that modulate specific downstream
S/G2 G1
target proteins that activate signaling pathways involved
in DNA repair and cell cycle arrest.87,88 Further, when
Figure 5-5. Regulation of cyclin B/cdc2 activity during the cell cycle.
cellular damage is irreparable, checkpoint signaling
As cells enter into G2 phase, cyclin B/cdc2 complexes form and are kept
could eliminate potentially hazardous cells by permanent
inactive by phosphorylation; they are activated by dephosphorylation at
the end of G2 to lead cells into mitosis. Phosphorylation of cdc2 is cell cycle arrest or apoptosis.The physiological relevance
carried out by Wee-1 kinase and a Wee-1 related kinase, Myt1.The
of these signaling pathways is supported by their evolu-
Cdc25C phosphatase counteracts Wee1 and Myt1 activity and is a
tionary conservation and the finding that the major conse-
positive regulator of cdc2. Dephosphorylation of cdc2 by Cdc25C in
quence of their alteration in humans is tumorigenesis.87,88
late G2 activates the cyclin B/cdc2 complex and initiates mitosis.The
cyclin B/cdc2 complex is thought to phosphorylate Cdc25C, which
further activates Cdc25C, inducing the full activation of cyclin B/cdc2
G1/S Checkpoint
by forming an autocatalytic feedback loop.At the end of mitosis, cyclin
B is degraded by the APC, and cdc2 remains inactive until cyclin B levels
G1 is a phase in which cells make critical decisions about
increase again during late S and early G2.
their fates, including the commitment to replicate DNA
and complete the cell division cycle. As discussed pre-
viously, if the cellular milieu is favorable for proliferation,
Key APC substrates are the mitotic A- and B-type cyclins. a decision to enter S phase is made at a restriction point.
Cyclin A is degraded in metaphase, whereas B-type cyclins In unstressed cells, this commitment to replicate DNA and
are degraded when cells enter anaphase.80,81 Cyclin B1 divide is irreversible until the next G1 phase. If DNA is
destruction starts as soon as the last chromosomes are damaged, however, the G1/S checkpoint is integral for
aligned on the metaphase plate and is complete by the preventing transition of cells into S phase and replication
end of metaphase.82 Another group of APC substrates are of the damaged DNA template.In fact,if checkpoint signal-
proteins that function as anaphase inhibitors. During G2, ing is activated in G1, it can delay cell cycle progression
sister chromatids are held together by proteins called even if cells have already passed the restriction point. Due
cohesins, which require inactivation by APC for anaphase to its essential and rate-limiting role in G1/S transition,
initiation.83,84 Overall, the APC regulates two different cyclin E/cdk2 is a key target for the DNA damage check-
steps in mitosis. First, sister chromatid separation is point.35 Progression through G1 can be halted at either the
triggered by destruction of the anaphase inhibitors, after restriction point, by inhibition of RB phosphorylation, or
which spindle disassembly and mitotic exit are initiated by closer to the S phase transition by inhibition of cyclin
the degradation of mitotic cyclins.These two steps allow E/cdk2 activity. The activation of the G1 and subsequent
the cell to couple the exit from mitosis to the prior checkpoints during the cell cycle relies on a distinct
completion of anaphase. network of signaling pathways that ultimately regulate the
activity of the key enzymes of the cell cycle, the cdks.
Central to activation of the G1/S cell cycle checkpoint
CELL CYCLE CHECKPOINTS and all those that follow is the ability of the cell to  sense
stress and activate the requisite signaling pathways. The
At key transitions during eukaryotic cell cycle progression, stress that is best studied in human cells is that induced
signaling pathways monitor the successful completion of by DNA damage. For the G1 checkpoint to be effective in
events in one phase of the cell cycle before proceeding to blocking cell entry into S phase within minutes of
the next phase.These regulatory pathways are commonly exposure of cells to DNA damaging agents, machinery
referred to as cell cycle checkpoints.85 In a broader must be in place within cells that is poised to act without
context, cell cycle checkpoints are signal transduction the time requirement of transcription and translation.
pathways that link the rate of cell cycle phase transitions Pathways that satisfy this requirement involve proteins
to the timely and accurate completion of prior, dependent that can  sense DNA lesions and transduce this signal
events. Checkpoint surveillance functions are not through phosphorylation to effectors that can signal
confined solely to nuclear events, as parameters such as rapidly to downstream targets.87,88 How DNA lesions or
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5 " Control of the Cell Cycle 7
Parent cell
Cyclin B/cdc2 activity
Interphase
PlK1 activity
Chromatin condenses into chromosomes
Changes in microtubular network, actin
microfilaments, nuclear lamina and histone
phosphorylation
Chromosomes align at metaphase plate
Prophase
Sister chromatids separate,
centromeres divide
Chromotin expands
Cytoplasm divides
APC
Metaphase
Anaphase
Telophase
Figure 5-6. Key stages of mitosis.As the parent cell enters prophase, the chromosomes condense and proteins bind the kinetochores, preparing for
spindle attachment. Upon nuclear envelope breakdown, the cell enters prometaphase, during which the mitotic spindle is formed and the
chromosomes attach to microtubules in the spindle via their kinetochores. Once attached, the chromosomes align along the metaphase plate in the
center of the spindle. During metaphase, all of the chromosomes are attached to microtubules via their kinetochores and are aligned at the
metaphase plate.At anaphase onset, the sister chromatids separate and move toward the poles of the spindle. During telophase, the parent cell is
divided into two daughter cells by cytokinesis.
blocks to DNA synthesis are  sensed is not well under- two effectors, kinases Chk2 and Chk1, are activated by
stood; however, the key transducer proteins are members phosphorylation in response to DNA damage and
of the DNA-dependent protein kinase-like family that replication disturbances.87,89 91 After exposure of cells to
includes human ataxia telangiectasia mutated (ATM) and ionizing radiation,ATM phosphorylates Chk2 regardless of
ATM-related (ATR).89 91 These proteins are required for all cell cycle position.94,95 In response, however, to ultraviolet
checkpoints that are engaged by altered DNA structure radiation or exposure of cells to agents that inhibit DNA
and/or DNA lesions and activate effector kinases by replication (e.g., hydroxyurea), ATR activates Chk1 by
phosphorylation. ATM was originally cloned as the gene phosphorylation.96
mutation in ataxia telangiectasia (AT), an inherited disease For cells in G1 that are past the restriction point and near
that is characterized by predisposition to cancer.92,93 ATM, the G1/S transition, a key target for checkpoint signaling is
ATR, and DNA-PK are thought to act at very early stages the cyclin E/cdk2 complex (Fig. 5-7). After exposure of
after the initiation of DNA damage. In human cells, cells to UV or IR, the level of Cdc25A phosphatase rapidly
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Figure 5-7. Model of G1/S
Genotoxic Stress
checkpoint signaling after
genotoxic stress. In response to
genotoxic stress, the ATM/ATR
P
P
P
signaling pathway is activated,
Activation Maintenance
ATM
leading to phosphorylation and
p53
Chk1/2
ATR
activation of Chk1 and Chk2
kinases and subsequent
P
phosphorylation of Cdc25A. Ub
cdk
Phosphorylated Cdc25A is
P
Cdc25A
P
targeted for ubiquitin-mediated
p21
cyclin
degradation, which prevents
Cdc25A
Degradation
cyclin E/cdk2 activation and S
phase transition.ATM/ATR also
Cdc25C
activate p53-dependent signaling
P
P P
that contributes to maintenance
of the G1 arrest by transactivation
P
P
P P
RB
Cdc25C
of the cdk inhibitor p21, which
RB
binds to cyclin/cdk complexes to cdk2 cdk2
reduce RB phosphorylation and
cyclin E cyclin E
E2F
thus prevent E2F from mediating
Cyclin E & cdk2
E2F
transcription of genes, the
Inactive Active synthesis blocked
protein products of which are
G1 arrest S phase
required for DNA replication and
entry
further transition through the cell
cycle.
decreases.35 Cdc25A removes the inhibitory phosphoryla- can phosphorylate key targets Cdc25A and p53 within
tion on cdk2 that is required for G1/S transition.After IR or minutes after DNA damage, the impact of the signaling
UV exposure, Cdc25A is rapidly phosphorylated by Chk2 pathways regulated by Cdc25A and p53 on cdk2 activity
and Chk1, respectively. Chk-mediated phosphorylation and G1/S blockage are separated in time, due to the
triggers accelerated turnover of Cdc25A and thus inhibi- dependence of p53 signaling on transcription and protein
tion of cdk2.97,98 An endpoint of this checkpoint signaling synthesis.
is inhibition of cdk2-dependent loading of Cdc45 onto the In addition to phosphorylation, protein-protein interac-
DNA prereplication complexes and thus inhibition of S tions can modulate p53 half-life. One of the interactions
phase.99 involves a protein encoded by the INK4a/ARF locus.
An integral target for checkpoint signaling in cells that Alternatively spliced transcripts arising from this locus
in G1 transition before the restriction point is the p53 encode two tumor suppressor proteins, p16INK4A and
tumor suppressor protein.100 In normal, nonstressed cells, p19ARF (ARF), which regulate the activities of RB and p53,
p53 protein is maintained at low steady-state levels and respectively.111,112 As discussed previously, p16INK4A
has a very short half-life.101 104 This half-life is a result of inhibits the activities of cyclin D-dependent kinases, cdk4
the rapid MDM2-mediated degradation of the protein after and cdk6. On the other hand, when present in the cell,
synthesis. The importance of MDM2 for maintenance of ARF protein binds to MDM2 and disrupts MDM2-mediated
appropriate p53 levels in vivo is highlighted by the fact degradation of p53, leading to p53 stabilization and a
that absence of MDM2 in knock-out mice results in early p53-dependent transcriptional response.113 116 ARF gene
embryonic lethality that is rescued by a dual knock-out of expression is induced by oncogenic stimuli such as viral
MDM2 and p53.105,106 oncoprotein expression or elevated levels of c-myc or
After exposure of cells to stress (including DNA damage ras.117 119 The biological importance of ARF in the
or oxidative stress), p53 phosphorylation changes, and activation of p53 signaling pathways is exemplified by
protein levels increase significantly (see Fig. 5-7).100 the finding that ARF-deficient mice develop spontaneous
Identification of the kinases that phosphorylate p53 after tumors and have accelerated tumor progression after
genotoxic stress provided key links between the trans- carcinogen exposure, similar to p53-null mice.120 These
ducers and effector kinases of checkpoint signaling and findings provide the molecular basis for the stabilization
the downstream target proteins such as p53. Upstream of p53 observed in cells after oncogenic stimulation and
transducers that are required for p53-mediated mainte- demonstrate that this signaling pathway is distinct from
nance of G1 checkpoint arrest are the same as those that activated by genotoxic stress.121 Although the p53-
required for activation of the checkpoint, namely the mediated induction of the cdk inhibitor p21WAF1/Cip1
ATM/ATR and Chk2/Chk1kinases. Phosphorylation leads contributes to ARF-induced growth arrest, ARF can
to increased levels and activity of p53 as a transcriptional prevent proliferation of p21WAF1/Cip1-null primary mouse
activator. Among the genes regulated by p53, the cdk embryo fibroblasts (MEFs), indicating that other ARF-
inhibitor p21WAF1/Cip1 plays a central role in G1 checkpoint inducible genes can compensate.122,123 ARF also can
by inhibiting cdks that are essential for entry into S inhibit proliferation of MEFs lacking both MDM2 and p53,
phase.107 110 Thus, although ATM/ATR-mediated signaling implying that ARF can interact with targets other than
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5 " Control of the Cell Cycle 9
MDM2.124 Consistent with these findings, mice lacking proteins phosphorylated by ATM that contribute to
ARF, p53, and MDM2 develop multiple and more arrest of cells in S phase are Nbs1, Brca1, SMC1, and
aggressive tumors than mice lacking either gene alone.124 FAncD2.133 139 Once these proteins are phosphorylated,
Thus,ARF is a major component of a regulatory pathway there is an immediate cessation of initiation of new repli-
stimulated by oncogenic signals culminating in both cation forks for approximately 90 minutes, after which
p53-dependent and -independent signaling. Although the time replication begins to resume.It is not yet known how
importance of this pathway has been established overtly phosphorylation of these proteins prevents initiation of
in experimental animal oncology, it still must be docu- new replication forks, and mechanistic linkage of these
mented further in human oncology to understand the full signaling molecules to the DNA replication machinery
biological significance of ARF as a tumor suppressor in remains a major gap in our knowledge. Nevertheless, the
human cells. importance of this process in cancer formation in humans
Human cells also have evolved additional mechanisms is suggested by the fact that many of these genes are
to prolong a G1 cell cycle checkpoint arrest. For example, mutated in familial cancer syndromes. For example, the
after exposure of keratinocytes and melanocytes to cancer susceptibility syndromes Ataxia-telangiectasia,
physiological doses of UV radiation, there is an increase of Nijmegen breakage syndrome, Fanconi s anemia, and
the cdk inhibitor p16INK4a.125 Such secondary maintenance familial breast/ovarian carcinoma syndrome are caused by
pathways act in a cell-type and stimulus-specific manner. inherited mutations in ATM, Nbs1, FAncD2, and Brca1,
Given the direct role that cdk inhibitors play in regulation respectively.
of the G1/S transition, it is not surprising that cdk inhibitor
function is often compromised in human tumors. The
G2 Checkpoint
p16INK4A gene is the frequent target of mutations that
ablate its function, including point mutations, promoter In addition to activation of the G1/S and S phase check-
methylation, or homozygous deletions.126 Likewise, many points, DNA damage also activates checkpoint arrest in G2
human breast cancers have reduced p27Kip1 protein to prevent the passage of DNA lesions to two daughter
expression or aberrant subcellular localization of the cells during mitosis. These DNA damage checkpoint
protein that has been correlated with more aggressive pathways all share common upstream signaling pathways
tumors.127 130 made up of the ATM/ATR transducer and Chk2/Chk1
effector kinases. The biochemical pathways involved in
the DNA damage-induced G2 arrest involve signaling
S Phase Checkpoint
cascades that converge to inhibit the activation of cdc2
If one of the major goals of cell cycle checkpoints is to through maintenance of tyrosine-15 phosphorylation.140
prevent the deleterious consequences of replicating By preventing dephosphorylation of this inhibitory site,
damaged DNA, the responses of cells that are already in cyclinB/cdc2 is not activated, and cells remain arrested in
S phase at the time of the DNA damage will be critical for the G2 phase of the cell cycle.Evidence for this is provided
optimal outcome of the cell. Because DNA replication is by the experimental use of cdc2 mutants that cannot be
ongoing in S-phase cells,these cells must respond virtually phosphorylated at tyrosine-15. When these mutants are
instantaneously to halt initiation of new replication forks expressed in human cells, the G2 delay induced by DNA
throughout S phase. In response to the introduction of damage is abrogated.141
DNA double-strand breaks, such as those introduced by Activation of the G2 checkpoint after genotoxic stress
ionizing irradiation, this instantaneous response is involves ATM-mediated phosphorylation and activation of
initiated by activation of the ATM protein kinase.89 It has the Chk1 and Chk2 kinases (Fig.5-9).142 144 Both Chk1 and
recently been demonstrated that just a few breaks in the Plk1 are proposed to play a key role in the G2 arrest
cell s genome results in instantaneous activation of ATM through targeting the cdc2-specific phosphatase, Cdc25C,
protein throughout the cell, thus providing a mechanism for phosphorylation after DNA damage.143,145,146 One
by which the cell can respond quickly and completely to working model is that Chk1-mediated phosphorylation
the presence of broken DNA.131 This activation appears to of Cdc25C on serine-216, after DNA damage, creates a
occur through some aspect of chromatin structure that is binding site for 14-3-3 proteins.145 Because the 14-3-3
altered by the presence of broken DNA and through proteins are found in the cytoplasm in human cells, it is
autophosphorylation of the ATM protein. For responses to proposed that 14-3-3 proteins prevent cell transition into
other types of DNA damage, such as base damage caused mitosis by sequestering Cdc25C in the cytoplasm.147 Such
by exposure to ultraviolet (UV) light or alkylating agents, nuclear export would separate the phosphatase from its
the ATR kinase, rather than the ATM kinase, appears to be substrate, cyclin B/cdc2. Recent studies in yeast, however,
important for initiating the relevant signal transduction suggest that other levels of regulation of Cdc25C besides
pathways.132 nuclear export might participate in the G2 arrest induced
Once ATM or ATR has been activated by the intro- by DNA damage.88 It is proposed that direct inhibition
duction of DNA damage, these protein kinases begin to of Cdc25 activity by Chk1 is sufficient for proficient
phosphorylate substrates to help the cell arrest cell cycle checkpoint regulation of Cdc25 and that Cdc25C might
progression or repair DNA (Fig. 5-8). As discussed pre- be inhibited by another upstream kinase, Plk1.146 The
viously, the phosphorylation of p53, mdm2, and Chk2 activity of Plk1 is inhibited in the G2 phase of human
by ATM following DNA damage contributes to the arrest tumor cells exposed to ionizing radiation, camptothecin,
of cells in G1 before the restriction point. Among the and doxorubicin. Further, expression of a mutant Plk1 in
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Figure 5-8. Schematic
IR
representation of the signal
transduction pathways initiated
S957 S966
by ATM after ionizing irradiation
S1981
(IR) and the functional roles of
ATM the ATM targets.After IR, the
ATM ?? SMC1
specific activity of the ATM
kinase increases, and it
Radiosensitivity
subsequently phosphorylates
T68
Chk2, p53, and mdm2 to initiate
S20
S15
S1423
the G1 arrest; Nbs1, FancD2,
chk2
Brca1, and SMC1 to initiate the
p53
S343 S-phase arrest; and Brca1and
?
Brca1
S395 S1387 hRad17 to cause a G2 arrest.
mdm2
SMC1 is the only target of ATM
hRad17
SMC1
nbs1
where mutation of the ATM
phosphorylation sites affects
p21 Mre11
S957
Rad50
S645
S222 radiosensitivity.
S635
S966
FAncD2
G1 checkpoint
G2 checkpoint
or
apoptosis
S-phase checkpoint
which residues necessary for Plk1 activation are altered, In addition to a role in G1/S checkpoint function, p53-
prevents Plk1 inactivation and leads to G2 override in cells mediated signaling plays an integral role in maintenance
treated with doxorubicin.148 Studies have shown that of the G2 checkpoint delay after activation of the check-
normal epithelial cells and fibroblasts undergo G2 arrest point. Both p53 and several of its downstream targets are
in response to Plk inactivation in the absence of DNA necessary to maintain a G2 arrest after DNA damage, and
damage. Clearly, more investigation is required for a tumor cells lacking these proteins enter into mitosis with
complete understanding of the role of Chk1 and Plk accelerated kinetics.149 p53 is believed to exert G2 check-
activity in G2 checkpoint function. Further, there is not point responses through transcriptional upregulation of
sufficient evidence at this time to rule out the role of other the downstream target genes p21, 14-3-3Ã, and GADD45
Cdc25 family members in the G2 checkpoint. (see Fig. 5-9). Similar to its regulation of the cyclin D1/
Figure 5-9. Model of G2/M
checkpoint signaling after
genotoxic stress. In response to
genotoxic stress, the ATM/ATR
signaling pathway is activated,
leading to phosphorylation and
activation of Chk1 and Chk2
kinases and subsequent
phosphorylation of Cdc25C. Plk1
can also phosphorylate Cdc25C.
Genotoxic Stress
Phosphorylated Cdc25C can be
sequestered in the cytoplasm by
14-3-3 proteins, preventing cyclin
P
P
P
14-3-3Ã
B/cdc2 activation and mitotic
Activation Maintenance
ATM
Plk1
p53 entry.ATM/ATR also activate
Chk1/2 cdc2
ATR
p53-dependent signaling that
Cytoplasmic
contributes to maintenance of the
sequestration
P
14-3-3
G2 arrest by upregulating the 14-
3-3Ã protein that sequesters cdk1
P
Cdc25C
P
cdk
cdc2
p21 in the cytoplasm.The p53 gene
GADD45
Cytoplasmic
also transactivates the cdk
Cdc25C
cyclin
Cyclin B
sequestration
inhibitor p21, which binds to
cyclin/cdk complexes to reduce
?
RB phosphorylation and
P
eventually prevents E2F from
P P
mediating synthesis of cyclin B
P
P
P P
Cdc25C
RB and cdc2.The p21 gene also
RB
cdk2 cdc2
directly binds and inhibits cyclin
B/cdk1 complexes to block
Cyclin B Cyclin B
E2F
mitotic entry. Upregulation of
Cyclin B & cdc2
E2F
GADD45, mediated by p53, also
Inactive Active synthesis blocked
can inhibit cyclin B/cdc2 activity
G2 arrest M phase
through direct binding of the
entry
GADD45 to the complex.
Brca1
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5 " Control of the Cell Cycle 11
cdk4,6 or cyclin E/cdk2 complexes at the G1/S check- suggesting that disruption of the spindle checkpoint
point, p21 can bind to and inhibit the cyclin B1/cdc2 could occur during tumor progression.163 Because aneu-
complex and inhibit cyclin-activated kinase-mediated ploidy is a shared feature of a majority of cancer cells,
cdc2 activation.150 The p53-dependent increase in 14-3-3Ã future studies could reveal that additional components of
modulates the subcellular localization of the cyclin B1/ the spindle checkpoint pathway frequently are altered in
Cdc2 complex, as the binding of 14-3-3Ã to cdc2 results tumors.
in retention of the kinase in the cytoplasm.151 Loss of Integral to cell cycle regulation is the proper coordi-
14-3-3Ã also results in abrogation of the DNA damage G2 nation of mitotic exit and subsequent S phase entry.After
checkpoint and premature mitotic entry.151 The p53- DNA synthesis, cells have a tetraploid (4N) DNA content
mediated GADD45-dependent G2 arrest is induced only that is reduced to a diploid (2N) DNA content in each
after specific types of DNA damage, as lymphocytes from daughter cell after successful completion of mitosis.Intact
GADD45 knockout mice failed to arrest after exposure to checkpoint pathways are needed to prevent the S phase
UV radiation but retained the G2 checkpoint initiated by entry of cells that have failed to properly segregate their
ionizing radiation.152 GADD45 can directly inhibit the chromosomes during mitosis. Cells with defective spindle
cyclin B1/cdc2 complex.153 In addition to direct inhibition checkpoint function can exit from mitosis with a 4N DNA
of the cyclin B1/cdc2 complex by p21, p53 signaling content.72 These cells can inappropriately continue to the
can also mediate a reduction of cyclin B1 and cdc2 next cell cycle division and, in the absence of a functional
levels.154 156 The reduced expression of cyclin B1/Cdc2 G1/S checkpoint, enter S phase with a 4N DNA content;
is mediated in part by p53-dependent transcriptional this process is known as endoreduplication. Endoredupli-
repression of the cyclin B1 and cdc2 promoters and is RB cation results in the generation of polyploid cells that is,
dependent.155 The importance of p53-dependent regula- cells with a 4N or greater DNA content after mitotic exit.
tion of cdc2 activity is exemplified by the findings that Cells that are RB-, p53-, p21-, or p16-deficient can endo-
constitutive activation of cyclin B1/cdc2 activity overrides reduplicate after microtubule inhibitor treatment.164 168
p53-mediated G2 arrest.157 Thus, human cells have evolved The G1 cell cycle regulators, however, do not directly
multiple signaling pathways to establish and maintain a regulate the mitotic arrest induced by microtubule
G2 arrest. inhibitors; rather, absence of these proteins allows
deregulated cdk2 activity, the precise control of which is
required for normal cells to maintain proper coupling of
Spindle Checkpoint
mitotic exit and S-phase entry.167,169 Thus, in addition to
The existence of the mitotic checkpoint could be of key playing a role in checkpoint function after DNA damage,
importance to trap damaged cells that have escaped the proteins that mediate the G1/S checkpoint through
prior checkpoints due to absence of functional upstream regulation of cdk2 activity also prevent inappropriate S-
sensor or transducer proteins that regulate multiple phase entry after an abnormal mitotic exit and are critical
checkpoint pathways throughout the cell cycle, such as to proper coordination of S phase and mitosis.
ATM or p53. The mitotic spindle checkpoint monitors
spindle microtubule structure, chromosome alignment on
CELL CYCLE DYSREGULATION
the spindle, and chromosome attachment to kinetochores
during mitosis (Fig. 5-10).158 The spindle checkpoint IN HUMAN CANCERS
delays the onset of chromosome segregation during
anaphase until any defects in the mitotic spindle are Molecular analysis of human tumors demonstrates that
corrected. Unattached kinetochores are thought to be the alterations in components of the cell cycle machinery
source of the checkpoint signal,and mechanical tension at and checkpoint signaling pathways occur in the majority
the kinetochore dictates whether the checkpoint is of human tumors (Table 5-1). This finding underscores
initiated or not.159 Activation of the spindle checkpoint how important maintenance of cell cycle control is in the
prevents mitotic progression through inhibition of the prevention of human cancer. Alterations in the cell cycle
anaphase-promoting complex activator, Cdc20.160 machinery that occur most frequently include loss or
Mediators of the spindle checkpoint pathway include mutation of the RB tumor suppressor, overexpression
the Mad2, Bub1, and Bub3 proteins.158 Mad2 localizes to of cyclins, cdks, and Cdc25 phosphatases, and loss of
the kinetochores during prometaphase until alignment expression cdk inhibitors.The most frequently altered cell
of the chromosomes occurs in metaphase and regulates cycle checkpoint signaling molecule is the p53 tumor
mitotic exit by interaction with components of the APC suppressor. Proteins that reside upstream of p53
machinery (such as Cdc20) that mediate anaphase (including ATM and Chk2) are also targeted for mutation
entry.72,161 Bub1 and Bub3 also localize to kinetochores in human tumors, and their discovery and analysis have
and regulate chromosome/kinetochore interactions, and greatly deepened our insight on DNA damage response
both are required for cell cycle arrest after disruption signaling pathways.
of microtubule dynamics during mitosis.72 Expression of Mutations that affect the RB signaling pathway have
a dominant-negative Bub1 in cells abrogated spindle been identified in the majority of human cancers.42 RB
checkpoint function, as cells failed to undergo apoptosis function is defective in many human cancers, including
and continued through the cell cycle despite mitotic retinoblastoma, breast, osteosarcoma, and lung.170 The RB
spindle disruption.162 Inactivating mutations in Bub1 have gene was the first tumor suppressor gene identified, and
been identified in human colon carcinoma cell lines, shortly after validation of the RB gene as the locus that
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Figure 5-10. The spindle checkpoint.
Improper chromosome alignment on
the mitotic spindle, disruption of
microtubule dynamics, or unattached
kinetochores can activate the spindle
checkpoint. Spindle checkpoint
signaling is mediated by the Bub1,
Bub3, BubR1, and Mad2 proteins, all
of which localize to kinetochores. A,
Intact spindle checkpoint signaling
induces either metaphase arrest
through inhibition of APC or
Unattached kinetochores
induction of apoptosis. B, Defective
Disruption of microtubules
APC
Metaphase Anaphase
spindle checkpoint function from
either loss of Bub1- and Bub3-
dependent signaling or abrogation of
Mad2/BubR1-mediated APC inhibition
can lead to aberrant mitotic exit and,
in the absence of a functional G1/S
BUB1 MAD2
checkpoint, to the generation of
BUB3 BUBR1
aneuploid cells.
Apoptosis
A
Unattached kinetochores
Disruption of microtubules
APC
Metaphase Anaphase
Aberrant
BUB1 MAD2
BUB3 BUBR1
mitotic exit
Aneuploid
cell
B
underlies the development of both familial and sporadic In breast cancer, loss of normal RB function due to
retinoblastoma, mutational inactivation of the RB gene mutation is associated with 20% of tumors.175 In the
was implicated in the etiology of lung cancer, with greater 80% of breast carcinomas in which RB gene mutation is
than 90% of small-cell lung cancers having defective not observed, alterations in components of the signaling
RB.171 173 The evidence linking alterations of RB activity pathways that regulate RB are frequently found, including
with human lung tumorigenesis is unequivocal. The cyclin D1 and cyclin E overexpression and cdk4 and
frequency of RB pathway inactivation in lung cancer is so cdk6 gene amplification.170,176,177 Nearly 50% of invasive
high that it is reasonable to propose that disruption of breast cancers have elevated cyclin D expression com-
this pathway (through the genetic or epigenetic targeting pared with surrounding normal breast epithelium, while
of one RB or upstream signaling components) is a require- transgenic mice with overexpression of human cyclin
ment for the genesis of lung cancer.174 It is important to D1 or cyclin E in mammary cells develop mammary
note that inactivation of the parallel and interconnecting adenocarcinomas.178 180 Similarly, cdk4 and cdk6 gene
p14ARF/p53 axis is also essential in functionally RB- amplification occur in breast cancers, sarcomas, gliomas,
deficient lung cells to bypass efficient apoptosis.116 and melanomas.181
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5 " Control of the Cell Cycle 13
lymphomas, mesotheliomas, and pancreatic cancers.181,183
INK4A INK4B
CHECKPOINTS AND TUMORIGENESIS In tumor types in which p16 and p15 are not
deleted, methylation of the gene locus leads to transcrip-
To ensure high-fidelity DNA replication and division,
tional repression and loss of gene expression. In some
mammalian cells have evolved checkpoint signaling
tumors, the hypermethylation-associated inactivation
pathways that execute several tasks: rapid induction of cell
affects both p16INK4a and p14ARF, which is encoded by an
cycle delay, activation of DNA repair, maintenance of cell
alternative reading frame of p16INK4a.184
cycle arrest until repair is complete, reinitiation of cell
Both Cdc25A and Cdc25B phosphatases are over-
cycle progression if repair occurred, or initiation of
expressed in more than 30% of primary breast tumors,
apoptosis if the damage was irreparable.The many
checkpoints during the cell cycle provide a fail-safe 40% to 60% of non small-cell lung cancers, 50% of head
mechanism by which cells are repaired or eliminated
and neck tumors, and a significant fraction of non-
through apoptosis before the damaged DNA is transferred
Hodgkin s lymphomas.185 187 Elevation of these oncogenic
to daughter cells.Although a given individual cell in the
phosphatases can result in increased activation of cdk and
human body would not benefit from undergoing
override of checkpoint arrest. Cdc25B overexpression in a
apoptosis, this outcome would be highly beneficial to the
transgenic mouse model system results in increased
outcome of the individual in the prevention of
susceptibility to carcinogen-induced mammary tumors.188
tumorigenesis.
p53 gene mutation is the most frequently observed
mutation in the majority of human tumors. The impor-
tance of p53-dependent signaling in tumor suppression is
underscored by the frequency of mutation in sporadic
Modifications of cdk inhibitors that are upstream tumors and the finding that germline mutations of the p53
regulators of RB activity are also commonly found in gene result in Li-Fraumeni syndrome, a highly penetrant
human tumors. The cdk inhibitor p27KIP1 is often familial cancer syndrome associated with significantly
aberrantly expressed in human breast cancer, and reduced increased rates of brain tumors, breast cancers, and
KIP1
p27 protein levels are correlated with more aggressive sarcomas.189,190 In human tumors that lack p53 gene
breast tumors.127,128 Likewise, decreased expression of mutation, p53 function may be disrupted by alterations in
the cdk inhibitor p57KIP2 is found in human bladder cellular proteins that modulate the levels, localization, and
cancers.182 Germline mutations in p16INK4A predispose biochemical activity of p53. For example, in some tumors
individuals to melanoma, while deletion of the p15INK4B with wild-type p53 alleles, MDM2 gene amplification
INK4A
and p16 genes is linked to the pathogenesis of occurs, resulting in MDM2 protein overexpression and
TABLE 5-1
Mutations of Cell Cycle Checkpoint Regulators in Human Tumors*
TUMORS ASSOCIATED WITH MUTATIONS HEREDITARY SYNDROMES ASSOCIATED
GENE/PROTEIN OR ALTERED EXPRESSION WITH GERMLINE MUTATIONS
ATM Breast carcinomas, lymphomas, leukemias Ataxia-telangiectasia
Bub1 Colorectal carcinomas NR
BRCA1 Breast and ovarian carcinoma Familial breast and ovarian cancer
Cdc25A Carcinomas of breast, lung, head and neck, and lymphoma NR
Cdc25B Carcinomas of breast, lung, head and neck, and lymphoma NR
Cdk4 Wide array of cancers NR
Cdk6 Wide array of cancers
Chk1 Colorectal and endometrial carcinomas NR
Chk2 Carcinomas of breast, lung, colon, urogenital tract, and testis Li-Fraumeni syndrome
Cyclin D1 Wide array of cancers NR
Cyclin D2 Lymphoma and carcinomas of the colon, testis and ovary NR
Cyclin D3 Lymphoma, pancreatic carcinoma NR
Cyclin E Wide array of cancers NR
MDM2 Soft tissue tumors, osteosarcomas, esophageal carcinomas NR
MRE11 Lymphoma Ataxia-telangiectasia-like disorder
NBS Lymphomas, leukemias Nijmegen breakage syndrome
p15INK4B Wide array of cancers NR
p16INK4A Wide array of cancers Familial melanoma
p27KIP1 Wide array of cancers NR
p53 Wide array of cancers Li-Fraumeni syndrome
p57KIP2 Bladder carcinomas NR
p130 Wide array of cancers NR
RB Wide array of cancers Familial retinoblastoma
NR, not reported.
*Only alterations that are present in >10% of primary tumors are represented.
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subsequent p53 inactivation.191 In human papillomavirus- either make tumor cells more sensitive or normal cells less
induced cervical carcinoma, the p53 gene is typically not sensitive to these agents. The former result should
mutated; however, the human papillomavirus E6 protein facilitate tumor cell kill,and the latter result should reduce
binds p53 and targets it for degradation, abrogating p53- normal tissue toxicity. For example, building upon the
dependent signaling.192 signal transduction pathways initiated by ionizing
Mutation in components of the DNA damage response radiation (see Fig. 5-8), numerous different proteins could
pathway also leads to enhanced tumorigenesis as theoretically be targeted to enhance the radiation
discussed previously. For example, ATM mutations occur sensitivity of a tumor cell. Because it is easier to conceive
in ataxia telangiectasia, a disorder in which patients have of ways to inhibit enzymes like kinases than to restore
increased sensitivity to radiation and an elevated incidence function to proteins with structural defects, the ATM
of leukemias, lymphomas, and breast cancer.92,193 ATM-null and Chk2 kinases provide tantalizing targets to alter
mice exhibit growth retardation, neurologic dysfunction, radiosensitivity. Screens for small molecule inhibitors of
infertility, defective T lymphocyte maturation, and these kinases are underway to develop such sensitizing
sensitivity to ionizing radiation.194,195 The majority of ATM- agents. It is recognized that if such inhibitors are given
deficient animals develop malignant lymphomas by four systemically, sensitization of normal tissues could be a
months of age, while ATM-/- fibroblasts have abnormal problem. But because therapeutic radiation can be
radiation checkpoint function after exposure to ionizing delivered locally either with external beam radiation or
radiation.194,195 The DNA double-strand break repair gene with brachytherapy (delivered either through seeds
MRE11 is mutated in individuals with an ataxia- or through antibody conjugates), it is easy to conceive of
telangiectasia-like disorder.196 Mutations of Chk2 and scenarios in which these irradiation treatments would
Chk1 also arise in human cancers. Chk2 mutations have become more effective tumoricidal approaches when
been reported in several cancers, including lung, while used in combination with small molecule inhibitors of
Chk1 mutations have been observed in human colon and these kinases. Similar concepts would apply to almost
endometrial cancers.197,198 In addition, heterozygous any molecular target involved in controlling cellular
alteration of Chk2 occurs in a subset of individuals with Li- responses to DNA damage. Alternatively, it is conceivable
Fraumeni syndrome that lack p53 gene mutations.199 that augmenting cellular responses to DNA damage in
These findings support the theory that in human tumors normal tissues could reduce the toxicities normally
where the p53 gene is intact, the function of the tumor associated with chemotherapy and radiation therapy.
suppressor might be disrupted by alterations in cellular
proteins that modulate the levels or activity of p53.
Targeting cdk Activity
Spindle checkpoint disruption has also been linked
to the pathogenesis of several human tumors. Bub1 Because cdk activity in particular cdk2 activity is
mutations have been identified in human colon carcinoma frequently elevated in human tumors, inhibition of cdk
cells, and Bub1 mutation facilitates the transformation of activity is a rational strategy for anticancer therapies.
cells lacking the breast cancer susceptibility gene, Conceptual and practical problems impact the develop-
BRCA2.163,200 Recent studies by Michel et al. demonstrate ment and introduction of such drugs for clinical use,
that Mad2 haplo-insufficiency results in significantly however. Cdk2 is active throughout the cell cycle and
elevated rates of lung tumor development in Mad2+/ plays multiple roles in progression through the cell cycle,
mice compared with age-matched wild-type mice.201 as described previously.Also, it is likely that these effects
differ among different tumor and normal tissues. As a
result, inhibition of cdk2 is likely to have highly complex
THERAPEUTIC MANIPULATION
effects. Nonetheless, numerous pharmacological inhibitors
OF CELL CYCLE CONTROLS of cdks have been developed, and several are in clinical
testing.202 Problems in development of inhibitors have
Research over the past two decades has shown that altera- been related to specificity of the agents and unpredictable
tions in cell cycle machinery and checkpoint signaling toxicity profiles. One of the first compounds to be tested,
lead to tumorigenesis. These findings have important flavopiridol, arrests cancer cells at the G1/S and G2/M
implications for the optimization of current therapeutic transitions through inhibition of cdk2, cdk4, and cdc2
regimens and for the selection of novel cell cycle targets kinase activity.203 Flavopiridol has potent antiproliferative
for the future development of anticancer agents. A leading activity against a variety of human cancer cell lines and
goal of cancer-based research is to identify compounds has produced favorable clinical responses in Phase I and
that will target key cell cycle controls in a highly selective Phase II studies of patients with renal, colorectal, gastric,
manner. lung, and esophageal carcinomas.204 206 Ongoing clinical
trials are evaluating flavopiridol in non-Hodgkin s
lymphoma and in breast and prostate cancers.207 The
Targeting DNA Damage Response Proteins
clinical tests of flavopiridol and related chemical cdk
Since many of the anticancer agents currently used inhibitors has spawned further research efforts to design
clinically target the DNA of the tumor cell, it seems mechanism-based cdk inhibitors through manipulation
reasonable to build upon the insights gained in recent of the phosphorylation and cyclin-binding sites of cdk
years of the molecular controls of cellular responses to proteins. The specificity of cdk inhibitors, however,
DNA damage to design novel approaches that would remains a limiting factor, as severe side effects were
Ch.005.qxd 3/2/04 9:01 AM Page 15
5 " Control of the Cell Cycle 15
experienced by patients in Phase I and Phase II studies. Preclinical studies have shown that inhibiting HDAC
Several studies demonstrate that flavopiridol binds and activity can induce cell cycle arrest or differentiation in a
inactivates cytosolic aldehyde dehydrogenase and glyco- significant fraction of tumor cell types.226 Thus, the design
gen phosphorylase and inhibits global transcription, of drugs to inhibit histone deacetylase activity has been
bringing into question flavopiridol s mechanism of pursued.These compounds increase the acetylation state
action.208 210 of the chromatin, alter chromatin structure, and modulate
Staurosporine is a nonspecific protein kinase inhibitor gene expression required for cell cycle arrest. Histone
that can override DNA damage-induced G2 delay in deacetylase inhibitors can trigger a G2 arrest in normal
response to ionizing radiation.211 The cytotoxicity of human cells; however, this G2 arrest fails to occur in a
staurosporine has limited its potential clinical efficacy, diverse range of human tumor cells and they undergo
however, leading to the development of staurosporine apoptosis.227 The histone deacetylase inhibitors FR901228
analogs with improved specificity and reduced cyto- and MS-27-275 have potent in vitro and in vivo anticancer
toxicity.212 One such staurosporine derivative,UCN-01,is a activity, and FR901228 has demonstrated efficacy against
cdk2 inhibitor but also a potent abrogator of the G2 cell T-cell lymphoma in clinical trials.228 230 Because subsets
cycle checkpoint, and it increases the cytotoxic effect of of HDACs bind and regulate specific sets of genes, it is
DNA-damaging agents in human tumor cells.213,214 UCN-01 likely that the identification of select HDAC inhibitors will
significantly inhibits the growth of a variety of human provide anticancer effects that are selective for given
tumors in mice xenograft tumor models and is currently genetic lesions in a tumor cell.
in Phase I clinical trials showing promising results.215 217
Preclinical studies have provided many mechanistic
insights to UCN-01 activity.Treatment of tumor cells with SUMMARY
UCN-01 results in Cdc25C activation, although these are
indirect effects of UCN-01 inhibition of upstream kinases, Over the past several decades, investigators have
including cdk2.218 UCN-01 inhibits Chk1; however, the uncovered a wealth of information about the proteins
related Chk2 kinase and the upstream ATM kinase are controlling cell growth and division in human cells.A key
refractory to inhibition by UCN-01.219 As is the case for finding is that loss of cell cycle checkpoints is a universal
other cdk inhibitors, a major limitation of UCN-01 is lack alteration identified in human cancer.231 Although
of specificity. numerous genetic alterations can result in loss of normal
New approaches are beginning to address the issue of checkpoints, the hope is that common strategies will be
chemical cdk inhibitor specificity through modification developed against a wide variety of cancers. Even though
of screening procedures.220,221 Also, several selective several of the currently used anticancer therapies target
cdk2 inhibitors are currently under development.202 This nonselective and non mechanism-based targets, their
avenue of research will continue to reveal novel com- effectiveness, albeit limited in many cases, is likely due to
pounds that will be more potent and selective than those the fact that they ultimately target cell cycle regulatory
currently available, and it will identify other potential or DNA damage response signaling pathways, the status
targets of cdk inhibitors that might be beneficial or of which is different in normal cells vs. tumor cells.
antagonistic to therapeutic strategies. Identifying all the components of the cellular machinery
that control the cell cycle both positively and negatively is
vital to the continued development of anticancer agents
Targeting Chromatin-Modifying Enzymes
that can preferentially eliminate cancer cells and minimize
Although many existing anticancer drugs target kinases the toxicity to normal tissues.The information generated
involved in cell cycle or checkpoint signaling pathways, by the genomic and proteomic approaches using eukaryo-
other agents are under development that can regulate tic model systems will continue to reveal new cell cycle
tumor cell cycle transit through modulation of enzymes regulatory molecules. As our understanding of cell cycle
that modify the acetylation state of the histones that are regulation and checkpoint signaling increases, the goal is
an important component of cellular chromatin. Recent to use this knowledge in the design of mechanism-based
studies have identified molecular interaction between the therapeutics that will bring anticancer therapy to a new
cell-cycle regulatory apparatus and proteins that regulate level.There can be little doubt of the value of targeting cell
histone acetylation and deacetylation. For example, RB cycle in drug discovery.
binds both E2F proteins and histone deacetylase (HDAC)
complexes.222,223 HDACs play an important role in RB
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