Chapter 15
The Cell Cycle
Vincent W. Yang
The cell cycle is a process in which a series of events
occur in a cell that eventually leads to the reproduc-
tion of two daughter cells. By traversing through the cell
cycle, a cell accomplishes two major tasks: duplication
of its genome and division into two cells. The cell cycle
is subjected to regulation by environmental cues, such as
the availability of nutrients (growth factors or mitogens),
and can be halted if damages to the DNA are detected to
allow sufficient time for repairing the damaged DNA.
Such regulation is critically important in a tissue such as
the gastrointestinal tract where epithelial cells undergo
multiple rounds of cell division that are carefully orches-
trated to achieve tissue homeostasis. In contrast, many dis-
ease processes, notably cancers, are often the consequence
of dysregulated cell cycle due to mutations in key mol-
ecules that control the cell cycle. Understanding how the
cell cycle is regulated is therefore essential to understand-
ing the normal and abnormal physiology of the gastroin-
testinal tract.
15.1  COMPONENTS OF THE CELL CYCLE
A typical eukaryotic cell cycle contains several distinct
phases that progress in an orderly fashion — a phase can-
not commence without completion of the previous phase.
The four phases of the cell cycle are G1 (G for gap), S
(synthesis), G2, and M (mitosis) (Figure 15.1). The G1,
S, and G2 phases collectively make up the interphase.
The DNA content of a cell in the G1 phase is 2N (N is the
number of chromosomes), also known as diploid, whereas
the DNA content of a cell in the G2 phase is 4N (tetra-
ploid). The DNA content of a cell in the S phase varies
between 2N and 4N, depending on the stage of replication
of the cell. The M phase is in turn comprised of two proc-
esses: mitosis, in which the cell’s chromosomes are equally
divided between the two daughter cells, and cytokinesis (or
cell division), in which the cytoplasm of the cell divides
in half to form two distinct daughter cells. Typically, the
amount of time required for a single cell cycle in actively
proliferating human cells in culture is 24 hours. Of these,
the M phase takes approximately one hour to complete and
interphase takes up the remaining 23 hours.
Physiology of the Gastrointestinal Tract, Two Volume Set. DOI: 10.1016/B978-0-12-382026-6.00015-4
© 2012 Elsevier Inc. All rights reserved.
In addition to the four phases of the cell cycle listed
earlier, one phase that lies outside the cell cycle is called
the G0 (0 for zero) phase (Figure 15.1). Cells in this phase
are in the resting phase, which is often the result of their
leaving the G1 phase of the cell cycle. Typically, a cell
enters the G0 phase if the environment is not conducive for
the progression of the cell cycle, as in the event of depri-
vation of essential nutrients or growth factors, or if a cell
has reached a fully differentiated state such as a hepato-
cyte or neuron. In these conditions, the cell is sometimes
referred to as being in a quiescent state. Additionally, a cell
can enter the G0 phase and become senescent due to DNA
damage or telomere attrition. This is often an alternative to
self-destruction of the damaged cell by apoptosis.
15.1.1  M Phase
The M phase consists of mitosis and cytokinesis. Mitosis
is the process in which DNA condenses into visible chro-
mosomes, which is followed by the separation of the
chromosomes into two identical sets. Cytokinesis can
be considered as the last phase of mitosis when the two
daughter cells separate, each with a nucleus and cytoplas-
mic organelles. Mitosis begins with nuclear membrane
breakdown followed by condensation of the chromo-
somes and separation of the centrosomes (prophase). This
is accompanied by the formation of mitotic spindles,
which are attached on one end, the centrosomes, and at
the other end, the kinetochore, a protein structure located
at or near the centromeres of mitotic chromosomes (pro-
metaphase). At this point, kinetochores that are unat-
tached to the mitotic spindles generate a “wait” signal that
delays the onset of anaphase until all chromosomes are
properly attached and aligned. This signal is also called
mitotic checkpoint or spindle assembly checkpoint (SAC;
see Section 15.3.4). Following prometaphase, chromo-
somes congregate at the equatorial plate (metaphase)
before separating to the opposite poles (anaphase). This
is followed by the formation of new nuclear membranes
around the daughter nuclei and uncoiling of the chromo-
somes (telophase), and finally into the formation of a
cleavage furrow that leads to the formation of two daughter
451
452 SECTION  |  I  Basic Cell Physiology, Genetics, and Growth of the GI Tract

FIGURE 15.1  The phases of the cell cycle.  The cell cycle begins in the G1 phase of a diploid cell (DNA content  2N; N is the number of chro-
mosomes). After DNA replication is completed in the S phase, the cell enters the G2 phase and has twice the amount of the DNA (4N) of the starting
cell. This is followed by mitosis (M) and cell division, which leads to the formation of two diploid daughter cells. Cells in either mitosis or cell divi-
sion (also called cytokinesis) are in the M phase, whereas those in the other three phases (G1, S, and G2) are in the interphase. The time in which a cell
spends in each phase varies among the cell type and is not drawn to scale.

216
FIGURE 15.2  The stages of mitosis.  The different phases of mitosis are illustrated. SAC, spindle assembly checkpoint. (Modified from with
permission.)

cells (cytokinesis). Figure 15.2 illustrates the various
stages of mitosis.
15.2  CONTROL OF THE CELL CYCLE
The organization of the cell cycle and its control sys-
tem are highly conserved among eukaryotic organisms.
Abundant information on how the cell cycle is regulated
in vertebrates was derived from earlier studies in yeast.1–5
These studies demonstrated that both cell cycle progres-
sion and cell division are driven by the sequential activa-
tion, and subsequent inactivation, of two key classes of
regulatory molecules, cyclins and cyclin-dependent kinases
(CDKs).6–18 Cyclins and CDKs form heterodimers with the
former serving as the regulatory subunits and the latter cat-
alytic subunits; CDKs are inactive in the absence of their
cyclin partners. Specific pairs of cyclin/CDK dimers func-
tion in specific phases of the cell cycle (Figure 15.3). For
example, the cyclin E-CDK2 complex becomes active late
in the G1 phase of the cell cycle and is responsible for the
transition from G1 into S phase. When activated by a bound
cyclin, CDKs phosphorylate a series of downstream target
proteins, either activating or inactivating them, which then
orchestrate the coordinated entry into the next phase of
Chapter  |  15  The Cell Cycle 453

FIGURE 15.3  The cell cycle is driven by cyclin/cyclin-dependent kinase (CDK) complexes.  The various pairs of cyclin/CDK complexes and
their respective positions in the cell cycle are shown.

FIGURE 15.4  Relative expression levels of cyclins in the cell cycle.  The relative protein levels of the four different cyclins in various phases of the
cell cycle are demonstrated. The letters in parentheses indicate the classes of cyclins. The levels of cyclin D often fluctuate, depending on whether or
not growth factors or mitogens are present. (R) indicates the restriction point.

the cell cycle. The identities of the target proteins depend

on the particular combination of cyclin-CDK complexes.
While CDKs are constitutively expressed throughout the
cell cycle, cyclins are synthesized (and destroyed) in spe-
cific stages of the cell cycle (hence the name cyclin), which
is often dependent upon various signaling molecules. This
cyclic nature of cyclin expression ensures that CDKs are
activated and inactivated in a precise manner, safeguarding
the orderly progression of the cell cycle (Figure 15.4).
15.2.1  Cyclins
Cyclins were named this because they undergo a cycle of
synthesis and degradation in each cell cycle. Depending
on the timing of their expression and functions in the cell
cycle, cyclins are divided into four classes. Three of these
classes, G1/S cyclins, S cyclins, and M cyclins, are directly
involved in the control of cell cycle events. The fourth
class, the G1 cyclins, controls the entry into the cell cycle
in response to extracellular growth factors or mitogens. In
454 SECTION  |  I  Basic Cell Physiology, Genetics, and Growth of the GI Tract
the G1 phase, growth factors are necessary to initiate and
maintain the proper transition to the S phase. Early in the
G1 phase, growth factors stimulate the synthesis of G1 cyc-
lins, represented by the cyclin D family of cyclins, which
activates CDK4/6 to induce synthesis of downstream
targets, one of which is cyclin E.19 The rise in cyclin E
(a G1/S cyclin) levels and activity of its partner, CDK2,
drive the cell past a restriction point (R in Figure 15.4) in
the cell cycle after which the cell is committed to proceed-
ing to DNA synthesis, even if the growth factors are with-
drawn. Subsequently, the S cyclins (represented by cyclin
A) and M cyclins (represented by cyclin B), are required
for the initiation of DNA replication and entry into mitosis,
respectively (Figure 15.4).
15.2.1.1  G1 Cyclins
The G1 cyclins are composed of the D-type cyclins that
include cyclins D1, D2, and D3.9,20,21 Along with their
partners, CDK4 and CDK6, the G1 cyclins act early in the
G1 phase of the cell cycle. The levels of G1 cyclin are low
in the G0 phase and increase progressively upon addition
of growth factors or mitogens to the cells.22,23 The mecha-
nisms by which mitogens or growth factors activate cyc-
lin D1 are complex and occur at both transcriptional and
post-transcriptional levels.19 At the transcriptional level,
induction of cyclin D1 by growth factors is dependent
on the Ras/Raf/mitogen-activated kinase kinase (MEK)/
extracellular signal-regulated kinase (ERK) signaling path-
way.24–27 Once synthesized, cyclin D1 protein has a short
half-life28 — its turnover is governed by ubiquitination
and proteasomal degradation, which in turn are depend-
ent on phosphorylation of cyclin D1 by glycogen synthase
kinase-3β (GSK-3β).29,30 Growth factors prevent cyclin D1
degradation by inhibiting GSK-3β-dependent phosphoryla-
tion of cyclin D1 through the Ras/phosphatidylinositol-
3-OH kinase (PI3K)/AKT pathway.30 It was subsequently
demonstrated that growth-factor-induced cyclin D1 gene
transcription by the MEK/ERK pathway and cyclin D1
protein stabilization by the PI3K/AKT pathway need to
occur in a sequential manner with the former occurring
early and the latter occurring late in the G1 phase in order
to drive the progression of the cell cycle.31
One of the key targets of an activated cyclin D-CDK4/6
complex is the retinoblastoma (RB) protein.32–34 RB is one
of three “pocket protein” families of cell cycle regulator
proteins — the other two are p107 and p130 — and has
a major role in restraining the transition between G1 and
S phases of the cell cycle.35 In the absence of mitogenic
stimuli, RB interacts with and inhibits the activity of the
transcription factor E2F.36–38 As E2F binding sites are
present in the promoters of many genes required for cell
cycle progression,39–42 the inhibition of E2F by RB pre-
vents entry into the cell cycle. In addition to physically
interacting with E2F, RB also recruits chromatin remod-
eling enzymes such as histone deacetylases (HDACs)43
that often serve as transcription co-repressors.44–47 Thus,
the binding of RB to E2F does not just simply inhibit E2F
activity. The RB–E2F complex binds to promoters and
actively represses transcription by blocking activity of the
surrounding enhancers on the promoter.48–51
The activity of RB is governed by phosphorylation
catalyzed by CDKs.52–54 RB contains 16 potential phos-
phorylation sites by CDKs and oscillates between hypo-
phosphorylated and hyperphosphorylated forms during the
cell cycle.34 The form that inhibits E2F is the hypophos-
phorylated form. CDKs, in complex with their cyclin part-
ners, phosphorylate the hypophosphorylated form of RB,
leading to the hyperphosphorylated form. At least three dif-
ferent cyclin–CDK complexes are known to phosphorylate
RB during the cell cycle — cyclin D-CDK4/6 acts early in
G1, cyclin E-CDK2 in late G1, and cyclin A-CDK2 in the
S phase.22 In this way, RB becomes sequentially phospho-
rylated in the cell cycle. It has been shown that successive
phosphorylations of RB by cyclin D-CDK4/6 and cyclin
E-CDK2 are necessary for the complete hyperphosphor-
ylation of RB.53 The hyperphosphorylation of RB prevents
its binding to E2F, thus releasing E2F from transcriptional
repression.19,34 Once liberated from RB, E2F, along with its
partner transcription factor DP,55–60 activate transcription of
genes that are involved in nucleotide metabolism and DNA
synthesis, thus allowing entry into the S phase.35,37,61–68 It
is of interest to note that one of the target genes of an active
E2F encodes cyclin E.69–71 It was also shown that phospho-
rylation of RB by cyclin D-CDK4/6 and cyclin E-CDK2
triggers sequential intramolecular interactions in RB that
progressively block RB functions as cells move through
G1.69 The complete hyperphosphorylation of RB by cyclin
D-CDK4/6 and cyclin E-CDK2 coincides with the R point
(Figure 15.4), beyond which the cell is committed to com-
pletion of the cell cycle.19
15.2.1.2  G1/S Cyclins
Cyclins E1 and E2 (collectively considered as cyclin E)
are the G1/S cyclins.9 Transcription of the cyclin E gene
is regulated by E2F,32 which, as described earlier, is acti-
vated due to cyclin D-CDK4/6-stimulated phosphorylation
of RB. The amounts of cyclin E protein and its associated
kinase (CDK2) activity are maximal in late G1 and early
S phases (Figure 15.4).72,73 Cyclin E-CDK2 completes RB
phosphorylation in the G1 phase and the transition from
cyclin D-CDK4/6 to cyclin E-CDK2 accounts for the loss
of dependency on growth factors. Cyclin E-CDK2 phos-
phorylates RB on different sites from cyclin D-CDK4/6,
and these kinases may differentially impact the interac-
tion between RB and E2Fs, HDACs, and other chromatin
remodeling proteins.34 In contrast to cyclin D-CDK4/6, the
Chapter  |  15  The Cell Cycle
functions of cyclin E-CDK2 are not limited to G1 control.
Thus, cyclin E-CDK2 phosphorylates other substrates that
are more directly involved in the control of DNA replica-
tion, centrosome duplication, replication origin licensing,
and firing.74 The timing of cyclin E-CDK2 activation and
its broader range of substrates suggest that cyclin E-CDK2
spans the interface between G1 regulation and core cell
cycle machinery during S phase.
In early S phase, cyclin E-CDK2 activity abruptly
ceases as a consequence of cyclin E degradation (Figure
15.4). This is mediated by phosphorylation by both
GSK-3β and CDK2, which target cyclin E for ubiquitina-
tion by the SCFFbw7 E3 ligase, leading it to proteasomal
degradation.75–77 Like cyclin D1,30 the involvement of
GSK-3β, an enzyme that is inhibited by the PI3K/AKT sig-
naling pathway, in regulating stability of cyclin E suggests
that cyclin E can be influenced by at least one mitogen-
dependent signaling cascade.
15.2.1.3  S Cyclins
The S cyclins include both cyclins A1 and A2. Whereas
cyclin A1 is restricted to the germ cell lineages, cyclin A2
is ubiquitously expressed in all cell types.78 Low levels of
cyclin A2 are first detected at the G1/S boundary. The lev-
els then rise steadily as cells begin to replicate their DNA
and do not decline until cyclin A is degraded in late G2
(Figure 15.4). In S phase, cyclin A and its partner, CDK2,
phosphorylate substrates that commence DNA replica-
tion from preformed replication initiation complexes.79–81
Cyclin A-CDK2 is also required to coordinate the end of S
phase with activation of the mitotic cyclin-CDKs.82
15.2.1.4  M Cyclins
During G2, A-type cyclins (the S cyclins) are degraded by
ubiquitin-mediated proteolysis, whereas B-type cyclins
(the M cyclins) are actively synthesized (Figure 15.4). As a
consequence, CDK1 (also known as Cdc2) binds to B-type
cyclins — an association required for the commencement
of mitosis. CDK1 preferentially binds to two main B-type
cyclins, cyclins B1 and B2. In contrast, the third iso-
form, cyclin B3, may have a function in the meiotic cell
cycle.83 Cyclin B-CDK1 regulates events during both the
G2/M transition and progression through mitosis.84 This is
accomplished by the phosphorylation of over 70 proteins
by the cyclin B-CDK1 complexes,8 although the number
of substrates could be much larger.85 Phosphorylation
of target proteins leads to numerous events that include
separation of centrosomes,86 condensation of chromo-
somes,87,88 breakdown of the nuclear lamina,89 and disas-
sembly of the Golgi apparatus,90 among others. Finally,
inactivation of the cyclin B-CDK1 complexes is required
for proper exit from mitosis and this inactivation is
455
achieved by the degradation of B-type cyclins by ubiqui-
tin-mediated proteolysis that is regulated by the anaphase-
promoting complex/cyclosome (APC/C).91–95
15.2.2  Cyclin-dependent Kinases
As described earlier, CDKs are the catalytic subunits of a
relatively large family of serine/threonine protein kinases
with a primary role in cell cycle progression. The mamma-
lian genome contains 11 genes encoding CDKs and 9 others
encoding CDK-like proteins with conserved structure.8 The
prototype CDK, CDK1, was first identified in yeasts and
designated as Cdc2 in Saccharomyces pombe or Cdc28 in
S. cerevisiae.96,97 The mammalian homolog of yeast Cdc2,
CDK1, was subsequently identified due to its ability to com-
plement the yeast mutants.98,99 Other mammalian CDKs
were then identified by a host of techniques including com-
plementation, homology probing, differential display, and
PCR amplification with degenerate primers.100–106 Again,
as described earlier, progression through the G1 phase of the
cell cycle requires at least three CDKs — CDK4, CDK6,
and CDK2 — which, along with their regulatory cyclins,
target the RB family of proteins that include RB, p107, and
p130.35,51,69 CDK2 is also important for S phase progression
and CDK1 for G2/M transition and mitosis.
The kinase activities of CDKs are regulated at multiple
levels, including interaction with their regulatory subunits
(cyclins), binding to negative regulators called CDK inhib-
itors (CKIs; see Section 15.2.4), phosphorylation-dephos-
phorylation, folding, and subcellular localization.23,107
Among these, phosphorylation-dephosphorylation can
have activating or inhibitory effects on CDK activity. For
example, active cyclin-CDK complexes need to be phos-
phorylated in the T-loop of the CDK subunit by CDK-
activating kinase (CAK), which contains a complex of
CDK7, cyclin H, and MAT1 (Figure 15.5).108–110 In con-
trast, cyclin-CDK complexes can be negatively regulated
by phosphorylation in adjacent threonine and tyrosine
residues of the CDK subunit by the dual-specificity pro-
tein kinases WEE1 and MYT1.111–117 Conversely, these
inhibitory phosphorylations can be reversed by the ability
of the dual-specificity CDC25 phosphatases (CDC25A,
CDC25B, and CDC25C) to dephosphorylate the same
threonine and tyrosine residues and thus act as positive
regulators of cyclin-CDK activity (Figure 15.5).118–120 If
both activating and inactivating phosphorylations exist in
the same molecule, and they result in an inactive kinase.
15.2.3  In Vivo Functions of Cyclins and
CDKs as Revealed by Gene Knockout in Mice
Numerous studies have investigated the in vivo functions of
cyclins and CDKs in knockout mice with the genes deleted
either singly or in combination. Because it is not the intent
456 SECTION  |  I  Basic Cell Physiology, Genetics, and Growth of the GI Tract
FIGURE 15.5  The regulatory mechanisms of cell cycle
CDKs.  CDKs require binding to their cyclin partners for activation of
kinase activity. The INK4 proteins inhibit CDK activity by directly bind-
ing to monomeric CDK4 or CDK6. In contrast, Cip and Kip inhibitors
inactivate CDKs by binding to cyclin-CDK complexes. Cyclin-CDK
complexes are activated by phosphorylation of the CDK subunit by
cyclin-dependent activating kinase (CAK) that contains three subunits:
CDK7, cyclin H (cycH), and MAT1. In contrast, cyclin-CDK com-
plexes are inhibited by phosphorylation in adjacent threonine and tyro-
sine residues by the dual-specificity protein kinases, WEE1 and MYT1.
These inhibitory phosphorylations can be reversed by the dual-specificity
CDC25 phosphatases that dephosphorylate the CDKs at the same amino
acid residues. Cyc, cyclin. (From 8 with permission.)
of this chapter to review all of the phenotypes of the knock-
out mice, the readers are referred to the many excellent
recent review articles that summarize the findings of these
studies.8–10,121–123 From these studies, it becomes clear that
most of the cyclins and CDKs, although previously con-
sidered essential for cell proliferation, have turned out to
be dispensable. Several compensatory mechanisms were
uncovered among cyclins and CDKs. The particularly unex-
pected finding is that CDK2, thought to be a master regula-
tor of the cell cycle, is dispensable for the regulation of the
cell cycle with both CDK4 and CDK1 covering CDK2’s
functions. In fact, CDK1 alone is able to drive the mamma-
lian cell cycle,124 indicating that the regulation of the mam-
malian cell cycle is highly conserved.
15.2.4  CDK Inhibitors
CKIs are proteins that constrain the activities of CDKs.
Two classes of CDK inhibitors have been described.23 The
first class includes the inhibitors of CDK4 (INK4) proteins.
Four such INK4 proteins have been identified: p16INK4a
(also known as CDK inhibitor 2A or CDKN2A),125 p15INK4b
(CDKN2B),126 p18INK4c (CDKN2C),127,128 and p19INK4d
(CDKN2D).128,129 INK4 proteins specifically bind to and
inhibit monomeric CDK4 and CDK6 proteins.130 The sec-
ond class of CKIs includes the CDK-interacting protein/
CDK-interacting protein (Cip/Kip) family of proteins,
which are more broadly acting than the INK4 family of
proteins and do so by binding to cyclin-CDK complexes.131
There are three members of the Cip/Kip family of CKIs:
p21Cip1 (also called CDK inhibitor 1A or CDKN1A),132–137
p27Kip1 (CDKN1B),138–140 and p57Kip2 (CDKN1C).141,142
Cip and Kip inhibitors block CDK activity by forming inac-
tive trimeric complexes (cyclin E-CDK2, cyclin A-CDK2,
cyclin B-CDK1, and possibly cyclin D-CDK4 and cyclin
D-CDK6),143–148 thus exerting a much broader effect on the
progression of the cell cycle. Figure 15.5 summarizes the
multiple regulatory mechanisms by which CDK activities
are regulated.
15.3  CHECKPOINTS
The cell cycle contains several checkpoints to monitor and
regulate its progression.149–165 Checkpoints are positioned
at specific locations in the cell cycle to allow verification
of phase processes and repair of DNA damage. A cell can-
not proceed from one phase to the next without satisfying
all of the checkpoint requirements. An important function
of many of the cell cycle checkpoints is to assess DNA
damage. Upon detection of DNA damage, the checkpoint
initiates a signal cascade to either arrest the cell cycle until
repairs are properly made, or if repairs are not possible, to
target the cell for destruction via apoptosis as a means to
maintain genomic integrity. In the cell cycle, there are three
specific checkpoints for damaged or incompletely repli-
cated DNA: G1/S, G2/M, and intra-S checkpoints. These
checkpoints are patrolled by some of the CKIs described
earlier. Another important and specific checkpoint occurs
in mitosis, the so-called mitotic checkpoint or SAC. This
checkpoint is designed to monitor proper alignment of
the chromosomes during mitosis. Anaphase cannot pro-
ceed unless this checkpoint is satisfied. The locations of
the various checkpoints in the cell cycle are illustrated in
Figure 15.6. The various cell cycle checkpoint pathways in
response to DNA damage are summarized in Figure 15.7.
15.3.1  G1/S Checkpoint
The G1/S checkpoint (also called the G1 checkpoint)
is located near the end of the G1 phase, just before the
entry into S phase (Figure 15.6). In mammalian cells, the
G1 checkpoint is the restriction or R point (Figure 15.4).
This is a point where cells typically arrest the cell cycle
Chapter  |  15  The Cell Cycle
if environmental conditions are unfavorable for cell divi-
sion, such as the presence of DNA damage or lack of
growth factors. The G1 checkpoint is controlled by both
the INK4 and Cip/Kip families of CKIs. INK4 proteins
specifically bind to CDK4 and CDK6 and inhibit their
activity.130 Enforced expression of INK4 proteins arrest
cells in the G1 phase in an RB-dependent manner.166,167
FIGURE 15.6  The cell cycle checkpoints.  The locations of the vari-
ous checkpoints (stop signs) and the primary defects the checkpoints are
designed to monitor are shown.
FIGURE 15.7  A simplified scheme of the cell cycle checkpoint pathways in response to DNA damage.  The black arrows indicate activating and
red lines indicate inhibitory actions.
457
Here CDK4 is redistributed from cyclin D-CDK4 com-
plexes to INK4-CDK4 complexes, and unbound D-type
cyclin is rapidly degraded by the ubiquitination-mediated
proteasomal pathway.29 Also, in early G1 phase, the cyc-
lin E-CDK2 and cyclin A-CDK2 complexes are inhib-
ited by bound p21Cip1 and p27Kip1.148 Additionally, cyclin
D-CDK4/6 complexes bind p21Cip1 and p27Kip1.168–170
Loss of D-type cyclins therefore prevents the titration of
p21Cip1 and p27Kip1 by cyclin D-CDK4/6 complexes away
from the cyclin E-CDK2 and cyclin A-CDK2 complexes.
As a result, there is complete inhibition of cyclin E-CDK2
and cyclin A-CDK2 activities, as well as RB phosphoryla-
tion, leading to the exit from the G1 phase.171 Conversely,
growth-induced or oncogenic-induced expression of cyclin
D allows its interaction with CDK4/6 by competing with
INK4 for binding. The binding of cyclin D-CDK4/6 com-
plexes to p21Cip1 and p27Kip1 releases the inhibitors from
the cyclin E-CDK2 and cyclin A-CDK2 complexes. This
unleashes cyclin E-CDK2 and cyclin A-CDK2 activity,
allowing further RB phosphorylation, exit from G1, and
entry into S phase.171
The G1/S checkpoint is activated upon detection of
DNA damage. The mammalian DNA-damage response is
a complex network, involving a multitude of proteins that
include “sensor” proteins that sense the damage and trans-
mit signals to “transducer” proteins, which in turn convey
the signals to numerous “effector” proteins implicated in
458 SECTION  |  I  Basic Cell Physiology, Genetics, and Growth of the GI Tract
specific cellular pathways, including DNA repair mecha-
nisms, cell-cycle checkpoints, cellular senescence, and
apoptosis.172–177 In response to DNA damage, signals ini-
tiated by the sensors rapidly transduce to the ataxia tel-
angiectasia, mutated (ATM) and ataxia telangiectasia and
Rad3-related (ATR) kinases, which phosphorylate a great
number of substrates.178–182 Among the substrates phos-
phorylated by activated ATM and ATR are the checkpoint
serine/threonine kinases, checkpoint kinase 1 (CHK1) and
checkpoint kinase 2 (CHK2). To prevent entry into S phase,
CHK1 and CHK2 phosphorylate the cell cycle regulatory
phosphatase CDC25A, leading to its ubiquitin-mediated
proteolysis.183–185 Inactivation of CDC25A leads to sus-
tained inhibitory phosphorylation of cyclin E-CDK2 com-
plexes, thus preventing G1/S transition (Figure 15.5).183
ATM/ATR and CHK1/CHK2 kinases also target the tumor
suppressor, p53.186–192 The phosphorylation of p53 by
ATM/ATR inhibits its association with MDM2, an E3 ubiq-
uitin ligase that normally functions to keep the p53 level
low, stabilizing the p53 protein.193 Subsequently, p53 tran-
scriptionally activates expression of the p21Cip1 gene,134
which in turn inhibits cyclin E-CDK2 and prevents G1/S
transition.133 Lastly, rapid degradation of cyclin D1 in
response to DNA damage occurs independent of p53.194
The reduced cyclin D1 protein decreases the amount of
cyclin D-CDK4/6 complexes, resulting in the redistribution
of p21Cip1 to cyclin E-CDK2 complexes, resulting in the
latter’s inactivation.23
15.3.2  G2/M Checkpoint
The G2/M checkpoint (also known as the G2 checkpoint)
prevents cells from initiating mitosis when they experi-
ence DNA damage while in G2, when they progress into
G2 with either unrepaired DNA sustained during the pre-
vious S or G1 phase, or when they possess incompletely
replicated DNA from S phase.195,196 The critical target
of the G2 checkpoint is the mitosis-promoting activity of
the cyclin B-CDK1 complexes, whose activation after
genotoxic stresses is inhibited by ATM/ATR and CHK1/
CHK2-mediated degradation of the CDC25 family of
phosphatases, which normally activate CDK1 at the G2/M
boundary.183,185,197 In addition, other regulators of CDC25
and cyclin B-CDK1, such as the polo-like kinases (PLKs),
are targeted by DNA damage-induced mechanisms.196
Finally, the maintenance of the G2 checkpoint is depend-
ent on the transcriptional programs regulated by p53, lead-
ing to an induction of cell cycle inhibitors such as p21Cip1,
growth arrest and DNA damage-inducible 45 (GADD45),
and 14-3-3σ proteins.196,198 These proteins cooperatively
inhibit cyclin B-CDK1 activity by directly binding to cyc-
lin B-CDK1 (p21Cip1), dissociating CDK1 from cyclin
B (GADD45), and sequestering CDK1 in the cytoplasm
(14-3-3σ), resulting in G2 arrest.198
15.3.3  Intra-S Checkpoint
While proliferating cells respond to genotoxic stresses by
activating checkpoint responses that impose durable cell
cycle arrest in G1 or G2, before entry into S phase or mito-
sis, respectively, cells that experience genotoxic stresses
during DNA replication can only delay their progression
through the S phase in a transient fashion. If the damage is
not repaired during the delay, cells exit S phase and arrest
upon reaching the G2 checkpoint. Nonetheless, the intra-S
checkpoint is important for the maintenance of genomic
stability as DNA replication is a vulnerable period of the
cell cycle in which errors occur both endogenously or are
introduced exogenously. When the intra-S checkpoint
is activated, both replication initiation and fork progres-
sion are inhibited, thus reducing the rate of DNA replica-
tion.199 Studies have revealed numerous proteins that are
involved in the control of intra-S checkpoint.199–204 Among
these, activation of ATM and ATR is necessary to initi-
ate intra-S checkpoint upon sensing DNA double-strand
breaks (DSBs).205–207 Activated ATM and ATR then acti-
vate CHK1 and CHK2 kinases,208–211 which phosphorylate
CDC25A, leading to its ubiquitin-proteosome-dependent
degradation.183–185 This results in persistent inhibitory
phosphorylation of CDK2 in the cyclin A-CDK2 com-
plexes, which in turn prevents firing of the replication
origins.212 A second independent pathway is involved in
slowing down the replication rate in cells that have suffered
DNA damage. This effect is mediated by ATM-dependent
phosphorylation of a cohesion protein and structural main-
tenance of chromosomes-1 (SMC-1).213–215 However, the
mechanism by which phosphorylated SMC-1 interferes
with DNA replication remains unknown.
15.3.4  Mitotic Checkpoint or Spindle
Assembly Checkpoint
Mitosis is the process in which a cell divides itself into
two halves, each with an identical set of chromosomes
(Figure 15.2). The central regulator of this process is the
mitotic checkpoint as SAC, which is a signaling mecha-
nism that arrests the progression of metaphase to anaphase
until all chromosomes are attached to the mitotic spin-
dles.94,156,163,216–219 This signal is akin to an “anaphase
wait” signal that is generated at the kinetochores of unat-
tached chromosomes and is extinguished once all kineto-
chores are properly attached to the spindles (Figure 15.2).
Thus, sister chromatids are separated only when they are
in a position to be equally distributed to the two daughter
cells. Accordingly, the mitotic checkpoint serves to prevent
chromosome missegregation.
The proteins that control mitotic checkpoint were origi-
nally identified by screens for mutations that bypassed
the ability of wild-type S. cerevisiae to arrest in mitosis
Chapter  |  15  The Cell Cycle
in the presence of spindle poisons.220,221 The genes iden-
tified include MAD (mitotic-arrest deficient), MAD1,
MAD2, MAD3 (BUBR1 in humans), and budding uninhib-
ited by benzimidazole 1 (BUB1).220,221 It was later found
that these genes are conserved in all eukaryotes.163,222,223
When activated, these SAC proteins target CDC20, which
is a cofactor of the ubiquitin ligase anaphase promoting
complex/cyclosome (APC/C).94,224–234 Specifically, SAC
inhibits the ability of CDC20 to activate APC/C-mediated
ubiquitination of two key substrates, cyclin B and securin,
thus preventing their degradation by the 26S proteosome.
Proteolysis of cyclin B inactivates CDK1, allowing the
cells to exit from mitosis.235–239 On the other hand, destruc-
tion of securin leads to activation of separase, which is
required to cleave the cohesion complex that holds sister
chromatids together in order to execute anaphase.239–245
Therefore, by keeping CDC20 in check, the SAC allows
the chromosomes to properly align on the metaphase plate
and attach to the two spindle poles through mitotic spin-
dles. Once the chromosomes are properly oriented, the
checkpoint is extinguished, relieving the mitotic arrest and
allowing anaphase to proceed.
15.4  PATHOLOGICAL CONSEQUENCES
OF CELL CYCLE DEREGULATION OR
DYSREGULATION
Because regulation of the cell cycle is central to the con-
trol of cell proliferation, it is not surprising that cancers
are often the result of deregulation or dysregulation of the
cell cycle (e.g., colorectal cancer). Recent genomic-scale
sequencing studies have identified numerous somatic muta-
tions in genes that possibly are involved in the formation of
cancer.246,247 Among these, some of the most highly ranked
“cancer genes” are either directly or indirectly involved
in the regulation of the cell cycle. Examples include p53,
adenomatous polyposis coli (APC), KRAS, F-box, and
WD40 domain protein 7 (FBXW7), and phosphatidylinosi-
tol 3-kinase, catalytic, α-subunit (PI3KCA).246,247 Here we
briefly review some of the common cancer genes with cell
cycle regulatory functions.
15.4.1  Retinoblastoma Tumor Suppressor
Gene
The RB tumor suppressor protein limits cell prolif-
eration by preventing entry into the S phase of the cell
cycle.33,34,38,61,248,249 RB achieves its inhibitory effect
by blocking the activity of E2F. Progression into S phase
occurs when the ability of RB to suppress E2F is disrupted
by the hyperphosphorylation of RB by cyclin D- and cyclin
E-dependent CDKs in the G1 phase of the cell cycle.38 The
INK4 family of CKIs, particularly p16INKa, directly inhib-
its activities of the cyclin D-dependent kinases, CDK4 and
459
CDK6, thus maintaining RB in its active, anti-proliferative
state.125 Functional disruption of the tumor suppressors
p16INKa and RB, or overexpression of the proto-oncogene
products cyclin D1 and CDK4, occur in many human can-
cers, which prompts the speculation that disabling the “RB
pathway” is an essential part of cancer formation.22,250–255
In addition to being causative in retinoblastoma, loss of RB
has been found in many other cancers. Similarly, inactivat-
ing mutations of 16INK4a have been identified in numerous
tumors.256,257 The often exclusive nature of mutations that
result in RB or p16INK4a loss, and/or cyclin D1 or CDK4
overexpression, suggest that each of these cell cycle regula-
tory genes exerts a critical function in the RB pathway of
cancer formation.254
15.4.2  p53 and INK4a/ARF Tumor
Suppressor Genes
The tumor suppressor p53 is mutated in more than 50%
of human cancers.258 It has been estimated that cancers
derived from over 50 human cell types or tissues contain
mutations in the p53 gene.259,260 p53 is a labile protein but
accumulates in response to cellular stresses from DNA
damage, hypoxia, or oncogenic activation.261,262 Upon
stabilization and activation, p53 initiates a transcriptional
program that triggers either cell cycle arrest or apopto-
sis.263–269 Among the p53-responsive genes are p21Cip1,
BCL2-associated X protein (BAX), and mouse double-
minute 2 homolog (MDM2). While p21Cip1 regulates pro-
gression of the cell cycle by inhibiting cyclin E-, A-, and
B-CDK2 complexes, BAX causes apoptosis.270,271 The
transcriptional induction of MDM2 by p53 is a negative
feedback mechanism as binding of MDM2, an E3 ubiquitin
ligase, to p53 induces ubiquitination of p53 and subsequent
degradation.272–276 MDM2, in turn, is negatively regulated
by the alternative reading frame (ARF) tumor suppressor
(p14ARF in humans and p19ARF in mice).255 ARF directly
associates with MDM2 to block its interaction with p53,
therefore stabilizing p53.277–280 Thus, disruption of the
ARF-MDM2-p53 signaling pathway is a common feature
in cancers. This is supported by the finding that MDM2 is
overexpressed in 5–10% of human cancers, whereas ARF
is silenced or deleted in many others.281–285 It is of inter-
est to note that ARF is derived from an alternative reading
frame in exon 2 of the INK4a gene.286 Loss of the INK4a/
ARF locus, therefore, predisposes many tumor types due
to the dual disruption of the RB-E2F and MDM2-p53
pathways.
15.4.3  hCDC4/FBXW7 Tumor
Suppressor Gene
Abundant evidence indicates that ubiquitin-dependent
proteolysis regulates many aspects of the cell cycle and
460 SECTION  |  I  Basic Cell Physiology, Genetics, and Growth of the GI Tract
that dysregulation of the ubiquitin-proteosome pathway
contributes to the formation of cancers.287–291 The proteo-
lytic regulation of cell division is primarily controlled by
two ubiquitin ligase systems, APC/C and SCF. The APC/C
ubiquitin ligase is a multimeric protein complex that regu-
lates chromosome segregation (see Section 15.3.4). SCF
ligase complexes are structurally similar to APC/C; com-
posed of an invariable core complex of SKP1, CUL1, and
RBX1; and associated with a variable member of the F-box
protein family that serves as the substrate recognition com-
ponent.234,292–298 Among the approximately 70 different
F-box proteins that were identified, the human homolog
of yeast CDC4 (hCDC), also known as FBXW7, has been
extensively characterized and implicated in human tum-
origenesis. For example, among the approximately 140
mutated “cancer genes” identified in a recent genomic
screen in colorectal cancers (CRCs), FBXW7 ranks fifth as
the most frequently mutated gene (after APC, KRAS, p53,
and PI3K).246
Human FBXW7 exists in three different isoforms,
α, β, and γ, each with a unique amino terminal end
fused to a common carboxyl-terminal.299 The interaction
between FBXW7 and its substrates depends on phospho-
rylation of the substrate within a motif called the CDC4-
phophodegredron (CPD).300 This feature enables FBXW7
to simultaneously regulate a host of substrates by ubiquiti-
nation. Among the many substrates for FBXW7, some are
critically involved in the regulation of the cell cycle such as
cyclin E, c-Myc, c-Jun, and Notch.301–304 Mutations in the
FBXW7 gene, therefore, lead to stabilization and elevated
levels of these substrates. It is no wonder that FBXW7 is
such a commonly mutated gene in human cancers.301,305
One of the best characterized substrates of FBXW7 is
cyclin E, which is essential for entry into S phase from G1
phase in the cell cycle. Cyclin E level is elevated or dys-
regulated in many human cancers, resulting in dysfunc-
tion of the cell cycle. There are many consequences of
cyclin E deregulation including genetic instability, centro-
some amplification, and fork collapse during DNA replica-
tion.81,306–314 Several studies subsequently identified cyclin
E as a substrate for FBXW7, which mediates the phospho-
rylation- and ubiquitination-dependent degradation of cyc-
lin E.315–317 Thus, it appears that tumorigenesis secondary
to cyclin E deregulation is linked to altered function of
FBXW7/hCDC4.
15.4.4  Chromosomal Instability and
Aneuploidy as Consequences of an
Aberrant Mitotic Checkpoint
Genetic instability has long been recognized as an inte-
gral part of human cancers.318 In CRC, there are two
major forms of genetic instability: microsatellite insta-
bility (MIN) and chromosomal instability (CIN). MIN
tumors have mutations in the DNA mismatch repair
(MMR) genes and account for approximately 15–20% of
sporadic CRCs.319,320 The remaining sporadic CRCs have
CIN, which are frequently aneuploid, that is, they exhibit
alterations in the number of chromosomes.321 It has been
suggested that CIN is the driving force for the formation
of aneuploidy and tumorigenesis.156,216,322–325 Although
there has not been a unified mechanism responsible for
CIN, defects in several cellular processes have been caus-
ally linked to its formation. These include chromosome
dynamics (e.g., chromosome condensation, segregation,
cohesion, and kinetochore-spindle interaction), centrosome
duplication, cell cycle checkpoints (including G1, S, G2
and the SACs), DNA damage repair pathway, and telomere
functions.156,158,216,326–330 Among these potential factors
contributing to CIN, the mitotic checkpoint is probably
the most important one since it is an essential part of the
cell cycle that ensures equal distribution of chromosomes
upon the conclusion of cell division. Studies in mice lack-
ing specific components of the mitotic checkpoint support
this view. Mice with genetically reduced levels of mitotic
checkpoint proteins including MAD1, MAD2, BUB1,
BUB3, BUBR1, and centromere protein E (CENP-E)
all have increased levels of aneuploidy and CIN, with
the eventual formation of tumors in some animals.331–340
Importantly, somatic mutations of many of the same genes
have been identified in human cancers,341–349 indicating
the importance of the mitotic checkpoint in maintaining
genomic integrity. Lastly, germline mutations in BUB1B
have been associated with the rare recessive condition of
mosaic variegated aneuploidy (MVA), which is character-
ized by growth retardation, microcephaly, and childhood
cancer with a mosaic pattern of chromosome gains and
losses.350–353
15.4.5  Adenomatous Polyposis Coli Tumor
Suppressor Gene
Germline mutations of the APC (which is different from
APC/C) tumor suppressor gene cause familial adeno-
matous polyposis (FAP), an autosomal dominant disor-
der characterized by the presence of numerous colonic
adenomas and colon cancer early in life.354–356 Somatic
mutations in the APC gene were subsequently found to be
present in the majority of sporadic CRCs.357 Its involve-
ment in tumor initiation leads to the hypothesis that APC
functions as a “gatekeeper” of colonic epithelial cell
proliferation and is responsible for the maintenance of
colonic epithelial cell renewal.357 A telling insight into the
function of APC came from the observation that it inter-
acts with β-catenin,358,359 which is implicated in regulat-
ing the Wnt signaling pathway of growth control.360–365
Particularly, Wnt signaling is now a well-established cen-
tral mechanism for regulating proliferation of intestinal
Chapter  |  15  The Cell Cycle
FIGURE 15.8  Schematic presentation of the Wnt signaling pathway.  (A) Wnt is absent and (B) Wnt is present. Abbreviations: β-cat, β-catenin;
Fz, Frizzled; LRP, lipoprotein receptor-related protein; Dsh, disheveled; CK1, casein kinase 1; GSK, glycogen synthase kinase-3; TCF, T-cell factor
(Modified from 373 with permission.)
epithelial stem cells, which are the precursors to all sub-
sequent intestinal epithelial cell lineages.366–372 Wnt sign-
aling is normally absent in a quiescent, non-cycling cell.
This is accompanied by the sequestration of β-catenin in a
“destruction complex” in the cytoplasm that includes APC,
Axin, CK1, and GSK-3 (Figure 15.8A).373 This complex
leads to the phosphorylation of β-catenin, which is then
degraded by ubiquitin-mediated proteasomal degrada-
tion.374–381 When Wnt, a secreted glycoprotein, is present,
it binds to the cell surface receptors Frizzled (Fz) and
lipoprotein receptor-related protein (LRP). This leads to
activation of the protein disheveled (Dsh) and subsequent
release of β-catenin from the destruction complex, result-
ing in accumulation of free β-catenin in the cytoplasm
(Figure 15.8B). Some of this free β-catenin is shuttled into
the nucleus where it becomes associated with the tran-
scription factor, T-cell factor (TCF), to activate target gene
expression (Figure 15.8B). Among the genes stimulated by
the β-catenin/TCF complexes are those encoding cyclin D1
and c-Myc,382–384 both of which are critical for the pro-
gression of the cell cycle. It is important to note that muta-
tional inactivation of APC or constitutional mutational
activation of β-catenin, two common events in the patho-
genesis of CRC, results in a similar net effect of nuclear
β-catenin accumulation to that seen during Wnt signaling.
These mutations then lead to unregulated cell proliferation.
APC has also been implicated in the regulation of
mitosis in a β-catenin-independent manner. Here, APC is
461
localized to the ends of microtubules embedded in kine-
tochores and forms a complex with several SAC proteins.
This finding suggests that APC is involved in chromosome
segregation.385–389 The role of APC in regulation of mito-
sis is consistent with the finding that mutations in the APC
gene cause chromosomal instability.390–395 Thus, APC
may fulfill two requirements for a colonic epithelial cell to
develop into colon cancer: the cell must acquire a selective
advantage to allow initial clonal expansion, and the cell
must generate genetic instability to allow multiple hits in
other genes that are responsible for tumor progression and
malignant transformation.
15.5  CONCLUSION
The cell cycle is a fundamental cellular process in which
a cell duplicates itself in a highly faithful manner to main-
tain genomic integrity. The cell cycle is divided into several
distinct phases that are controlled by specific pairs of cyc-
lins and CDKs. The cell cycle also contains specific check-
points with which to monitor the presence of damaged or
incompletely replicated DNA in interphase or unaligned
chromosomes in mitosis. Dysregulation or deregulation of
the cell cycle often has pathological consequences and a
good example is cancer. Hence, many of the cell cycle reg-
ulators act as tumor suppressors or oncoproteins, such as
Rb, p53, FBXW7, and cyclins. The relevance of cell cycle
regulation also has an impact on the physiology of the
462 SECTION  |  I  Basic Cell Physiology, Genetics, and Growth of the GI Tract
gastrointestinal tract because the epithelium is composed
of rapidly proliferating cells. A prominent example is the
crucial role of the Wnt signaling pathway in regulating pro-
liferation of intestinal stem cells, and that a critical com-
ponent of the Wnt pathway, the APC protein, is mutated in
the majority of CRCs. Thus, a clear understanding of the
mechanisms regulating cell cycle progression is essential
for understanding the regulation of proliferation and differ-
entiation of intestinal epithelial cells.
ACKNOWLEDGMENT
The author wishes to thank Bing Yu for helping with some of the fig-
ures. This work was in part supported by grants from the National
Institutes of Health (DK052230 and CA084197).
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Chapter  |  15  The Cell Cycle
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