The document provides an overview of the cell cycle, detailing its phases (G1, S, G2, M), regulatory mechanisms involving cyclins and cyclin-dependent kinases (CDKs), and the consequences of dysregulation. It emphasizes the importance of cyclin-CDK complexes in regulating transitions between phases, particularly in response to growth factors. Additionally, it discusses the involvement of checkpoints in monitoring cell cycle progression and ensuring DNA damage is addressed or leads to apoptosis if necessary.

2. CELL CYCLE: Phases &control and
analysis
By
Romissaa Aly
Lecturer of Oral Medicine, Periodontology, Diagnosis
and Dental Radiology (Al-Azhar University)

3. • Contents:
• COMPONENTS OF THE CELL CYCLE
• CONTROL OF THE CELL CYCLE
• PATHOLOGICAL CONSEQUENCES OF CELL CYCLE
DEREGULATION OR DYSREGULATION
• Quantitative analysis of cell cycle phase durations

4. COMPONENTS OF THE CELL CYCLE

5. A typical eukaryotic cell cycle
contains several distinct
phases, which progress in an
orderly fashion—a phase
cannot commence without
completion of the previous
one.
The four phases of the cell
cycle are G1 (G for gap), S
(synthesis), G2, and M
(mitosis) phases (Fig. 8.1).
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
(tetraploid).
The DNA content of a cell in
the S phase varies between
2N and 4N, depending on
the stage of replication.
The M phase is in turn comprised of
two processes: 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 1 hour to complete and
interphase takes up the remaining 23
hours

6. In addition to the four
phases of the cell cycle
listed above, one phase
that lies outside the cell
cycle is called the G0 (0
for zero) phase.
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 G0
phase if the environment
is not conducive for the
progression of the cell
cycle, as in the event of
deprivation of essential
nutrients or growth
factors, or if a cell has
reached a fully
differentiated state such
as a hepatocytes or
neuron.
In these conditions, the
cell is sometimes referred
to as 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.

7. In the M phase,
mitosis commences
when DNA
condenses into
visible
chromosomes,
followed by the
separation of the
chromosomes into
two identical sets.
Cytokinesis is the
last phase of mitosis
when the two
daughter cells
separate, each with
a nucleus and
cytoplasmic
organelles.
Mitosis begins with
nuclear membrane
breakdown followed
by condensation of
the chromosomes
and separation of
the centrosomes
(prophase).
This is accompanied
by the formation of
mitotic spindles,
which are attached
on one end, the
centrosomes, and
the other end,
kinetochore, a
protein structure
located at or near
the centromeres of
mitotic
chromosomes
(prometaphase).
At this point,
kinetochores that
are unattached 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),
which is satisfied
once all
chromosomes are
congregated at the
equatorial plate
(metaphase).

8. This is followed by separation of the chromosomes to the opposite poles (anaphase) and
formation of new nuclear membranes around the daughter nuclei and uncoiling of the
chromosomes (telophase), eventually forming a cleavage furrow that leads to the formation of
two daughter cells (cytokinesis). Fig. 8.2 illustrates the various stages of mitosis

11. CONTROL OF THE CELL CYCLE

12. The organization of the
cell cycle and its control
system are highly
conserved among
eukaryotic organisms.
Abundant information
on how the cell cycle is
regulated in vertebrates
was derived from
earlier studies in
yeast.1–4
These studies demonstrate
that both cell cycle
progression and cell division
are driven by the sequential
activation, and subsequent
inactivation, of two key
classes of regulatory
molecules, cyclins, and
cyclin-dependent kinases
(CDKs)5–11 Cyclins and CDKs
form heterodimers with the
former acting as the
regulatory subunits and the
latter catalytic subunits;
CDKs are inactive in the
absence of their cyclin
partners.
Specific pairs of cyclin-
CDK dimers function in
specific phases of the
cell cycle

13. For example, the cyclin E-CDK2 complex becomes active late in the G1 phase 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 the cell cycle.
The identities of the target proteins depend on the combination of cyclin-CDK complexes.
While CDKs are constitutively expressed throughout the cell cycle, cyclins are synthesized (and destroyed) in specific
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 and
safeguards the orderly progression of the cell cycle.

15. 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
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 Fig. 8.4) in the
cell cycle after which the cell is irreversibly committed to proceeding to DNA synthesis, even if the growth factors are withdrawn.
. Early in the G1 phase, growth factors stimulate the synthesis of G1 cyclins, represented by the cyclin D family of cyclins, which
activates CDK4/6 to induce synthesis of downstream targets, one of which is cyclin E.12,13
The fourth class, the G1 cyclins, controls the entry into the cell cycle in response to extracellular growth factors or mitogens. In
the G1 phase, growth factors are necessary to initiate and maintain the proper transition to the S phase
Three of these classes, the G1/S cyclins, S cyclins, and M cyclins are directly involved in the control of cell cycle events.

17. The G1 cyclins are composed of the
D-type cyclins that include cyclins
D1, D2, and D3.7 Along with their
partners, CDK4 and CDK6, G1
cyclins act early in the G1 phase of
the cell cycle.
The levels of G1 cyclin are low in
G0 phase and increase
progressively upon addition of
growth factors or mitogens to the
cells.
The mechanisms by which
mitogens or growth factors activate
cyclin D1 are complex and occur at
both transcriptional and
posttranscriptional levels.
At the transcriptional level,
induction of cyclin D1 by growth
factors is dependent on the
RAS/RAF/mitogen activated kinase
(MEK)/extracellular signal-
regulated kinase (ERK) signaling
pathway.
Once synthesized, cyclin D1 protein has a short half-
life20—its turnover being governed by
ubiquitination and proteasomal degradation, which
in turn are dependent on phosphorylation of cyclin
D1 by glycogen synthase kinase-3β (GSK-3β).21,22
Growth factors prevent cyclin D1 degradation by
inhibiting GSK3β-dependent phosphorylation of
cyclin D1 through the Ras/phosphatidylinositol-3-OH
kinase (PI3K)/AKT pathway

18. One of the key targets of an
activated cyclin D-CDK4/6
complex is the
retinoblastoma (RB) protein.
RB is one of three “pocket
protein” family of cell cycle
regulator proteins—the
other two being p107 and
p130—and has a major role
in restraining the transition
between G1 and S phases of
the cell cycle.
In the absence of mitogenic
stimuli, RB interacts with and
inhibits the activity of the
transcription factor E2F.28–30 As
E2F-binding sites are present in
the promoters of many genes
required for cell cycle
progression,31–34 the inhibition
of E2F by RB prevents entry into
the cell cycle.
In addition to physically interacting with
E2F, RB also recruits chromatin
remodeling enzymes such as histone
deacetylases (HDACs)35 that often serve
as transcription corepressors.36–39 Thus,
the binding of RB to E2F not only simply
inhibits E2F activity, but the RB-E2F
complex binds to promoters and actively
represses transcription by blocking
activity of the surrounding enhancers on
the promoter.

19. The activity of RB is
governed by
phosphorylation catalyzed
by CDKs. RB contains
potential phosphorylation
sites by CDKs and oscillates
between
hypophosphorylated and
hyperphosphorylated forms
during the cell cycle.
The form that inhibits E2F is
the hypophosphorylated
form. CDKs, in complex with
their cyclin partners,
phosphorylate the
hypophosphorylated form of
RB, leading to the
hyperphosphorylated form.
At least three different
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.
In this way, RB becomes
sequentially phosphorylated
in the cell cycle. It has been
shown that successive
phosphorylation of RB by
cyclin D-CDK4/6 and cyclin
E-CDK2 is necessary for the
complete
hyperphosphorylation of RB

20. Cyclins E1 and E2
(collectively
considered as cyclin E)
are the G1/S cyclins.7
Transcription of the
cyclin E gene is
regulated by E2F,24,67
which, as described
above, is activated
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 .
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
phosphorylates RB on
different sites from
cyclin D-CDK4/6, and
these kinases may
differentially impact
the interaction
between RB and E2Fs,
HDACs, and other
chromatin remodeling
proteins.26 In contrast
to cyclin D-CDK4/6,
the 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
replication,
centrosome
duplication,
replication origin
licensing and firing.
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.

21. The S cyclins
include both
cyclins A1 and
A2.
While cyclin A1 is restricted
to the germ cell lineages,
cyclin A2 is ubiquitously
expressed in all cell types.
Low levels of cyclin A2 are first
detected at the G1/S boundary.
The levels then rise steadily as cells
begin to replicate their DNA and do
not decline until cyclin A is degraded
in late G2 (Fig. 8.4).
In S phase, cyclin A and its partner, CDK2,
phosphorylate substrates that commence DNA
replication from preformed replication initiation
complexes. Cyclin A-CDK2 are also required to
coordinate the end of the S phase with
activation of the mitotic cyclin-CDKs.

22. During G2, A-type cyclins (the S
cyclins) are degraded by
ubiquitin-mediated proteolysis
whereas B-type cyclins (the M
cyclins) are actively synthesized
(Fig. 8.4).
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
isoforms, cyclin B3, may have a
function in the meiotic cell cycle.
Cyclin B-CDK1 regulate events
during both the G2/M transition
and progression through mitosis.
This is accomplished by the
phosphorylation of over 70
proteins by the cyclin B-CDK1
complexes 6 although the
number of substrates could be
much larger.
Phosphorylation of target
proteins leads to numerous
events that include separation of
centrosomes.
condensation of chromosomes,
breakdown of the nuclear lamina,
and disassembly of the Golgi
apparatus, among others.
Finally, inactivation of the cyclin B-
CDK1 complexes is required for
proper exit from mitosis and this
inactivation is achieved by the
degradation of B-type cyclins by
ubiquitin-mediated proteolysis that
is regulated by the anaphase-
promoting complex/cyclosome
(APC/C).

23. CDKs are the
catalytic
subunits of a
relatively large
family of
serine/threonin
e protein
kinases with a
primary role in
cell cycle
progression.
The mammalian
genome contains 11
genes encoding
CDKs and nine
others encoding
CDK-like proteins
with conserved
structure.
The prototype CDK,
CDK1, was first
identified in yeasts and
designated as Cdc2 in
Saccharomyces pombe
or Cdc28 in
Saccharomyces
cerevisiae.
The mammalian
homologue of yeast
Cdc2, CDK1, was
subsequently
identified due to its
ability to complement
the yeast mutants.
Other mammalian CDKs
were then identified by
a host of techniques
including
complementation,
homology probing,
differential display, and
PCR amplification with
degenerate primers.

24. In contrast, cyclin-CDK complexes can be
negatively regulated by phosphorylation in
adjacent threonine and tyrosine residues of
the CDK subunit by the dual-specificity
protein kinases WEE1 and MYT1.
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.
If both activating and inactivating
phosphorylations exist in the same
molecule, they result in an inactive kinase.

26. Mammalian cyclin-dependent kinases. Trends Biochem Sci 2005;30(11):630–41, with permission from Elsevier).
These inhibitory phosphorylations can be reversed by the dual-specificity CDC25 phosphatases that dephosphorylate the CDKs at the same
amino acid residues. Cyc is cyclin. (Reproduced from reference Malumbres M, Barbacid M.
By contrast, cyclin-CDK complexes are inhibited by phosphorylation in adjacent threonine and tyrosine residues by the dual-specificity
protein kinases, WEE1 and MYT1.
Cyclin-CDK complexes are activated by phosphorylation of the CDK subunit by cyclin-dependent activating kinase (CAK) that contains three
subunits: CDK7, cyclin H, and MAT1.
The INK4 proteins inhibit CDK activity by directly binding to monomeric CDK4 or CDK6. In contrast, Cip and Kip inhibitors inactivate CDKs by
binding to cyclin-CDK complexes.
FIG. 8.5 The regulatory mechanisms of cell cycle CDKs. CDKs require binding to their cyclin partners for activation of kinase activity.

27. CDK inhibitors (CKIs) are proteins
that constrain the activities of CDKs.
Two classes of CDK inhibitors have
been described.15
The first class includes the INK4
proteins (inhibitors of CDK4). Four
such INK4 proteins have been
identified: p16INK4a (also known as
CDK inhibitor 2A or CDKN2A),129
p15INK4b (CDKN2B),130 p18INK4c
(CDKN2C),131,132 and p19INK4d
(CDKN2D).132,133
INK4 proteins specifically bind to and
inhibit monomeric CDK4 and CDK6
proteins.134
The second class of CKIs includes the
Cip/Kip (CDK-interacting
protein/CDK-interacting protein)
family of proteins which are more
broadly acting than the INK4 family
of proteins and do so by binding to
cyclin-CDK complexes.135
There are three members of the
Cip/Kip family of CKIs: p21Cip1 (also
called CDK inhibitor 1A or
CDKN1A),136–141 p27Kip1
(CDKN1B),142–144 and p57Kip2
(CDKN1C).145,146
Cip and Kip inhibitors block CDK
activity by forming inactive trimeric
complexes (cyclin E-CDK2, cyclin A-
CDK2, cyclin B-CDK1, and possibly
cyclin D-CDK4 and cyclin D-
CDK6),147–152 thus exerting a much
broader effect on the progression of
the cell cycle.

28. The cell cycle contains several
checkpoints to monitor and
regulate its progression.153–
169
Checkpoints are positioned at
specific locations in the cell
cycle to allow verification of
phase processes and repair of
DNA damage.
A cell cannot 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
replicated DNA: G1/S, G2/M,
and intra-S checkpoints.
These checkpoints are patrolled
by some of the CKIs described
above. A fourth 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
proceed unless this checkpoint
is satisfied

31. 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 (Fig. 8.6).
In mammalian cells, the G1 checkpoint is the restriction or R point (Fig. 8.4).
This is a point where cells typically arrest the cell cycle if environmental conditions are unfavorable for cell division, 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.134
Enforced expression of INK4 proteins arrest cells in the G1 phase in an RB-dependent manner.170,171
Here CDK4 is redistributed from cyclin D-CDK4 complexes to INK4-CDK4 complexes, and unbound D-type cyclin is rapidly
degraded by ubiquitination mediated proteasomal pathway.21
Also, in early G1 phase, the cyclin E-CDK2 and cyclin A-CDK2 complexes are inhibited by bound p21Cip1 and p27Kip1. 152 In
addition, cyclin D-CDK4/6 complexes bind p21Cip1 and p27Kip1. 172–174
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.

32. 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 transmit signals to “transducer”
proteins, which, in turn, convey the
signals to numerous “effector” proteins
implicated in specific cellular pathways,
including DNA repair mechanisms, cell
cycle checkpoints, cellular senescence,
and apoptosis.176–181
In response to DNA damage, signals
initiated by the sensors rapidly
transduce to the ATM (ataxia
telangiectasia, mutated) and ATR
(ataxia telangiectasia and Rad3-
related) kinases, which
phosphorylate a great number of
substrates.182–188
Among the substrates
phosphorylated by activated ATM
and ATR are the checkpoint serine/
threonine kinases, CHK1 (checkpoint
kinase 1) and CHK2 (checkpoint
kinase 2).
To prevent entry into S phase, CHK1
and CHK2 phosphorylate the cell
cycle regulatory phosphatase
CDC25A, leading to its ubiquitin-
mediated proteolysis.189–191
Inactivation of CDC25A leads to
sustained inhibitory phosphorylation
of cyclin E-CDK2 complexes, thus
preventing G1/S transition189
(Fig. 8.5).

33. The G2/M checkpoint
(also known as G2
checkpoint) prevents
cells from initiating
mitosis when they
experience DNA damage
while in G2, when they
progress into G2 with
either unrepaired DNA
sustained during the
previous S or G1 phase,
or when they possess
incompletely replicated
DNA from S
phase.207,208
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,
CHK1/CHK2-mediated
degradation of CDC25
family of phosphatases,
which normally activate
CDK1 at the G2/M
boundary.189,191,209
In addition, other
regulators of CDC25 and
cyclin B-CDK1, such as
the Pololike kinases
(PLKs) are targeted by
DNA damage-induced
mechanisms.208
Finally, the maintenance
of the G2 checkpoint is
dependent on the
transcriptional programs
regulated by p53, leading
to an induction of cell
cycle inhibitors such as
p21Cip1, growth arrest
and DNA damage-
inducible 45 (GADD45),
and 14-3-3σ
proteins.208,210
These proteins
cooperatively inhibit
cyclin B-CDK1 activity by
directly binding to cyclin
B-CDK1 (p21Cip1),
dissociating CDK1 from
cyclin B (GADD45), and
sequestering CDK1 in the
cytoplasm (14-3-3σ),
resulting in G2 arrest.

34. Mitosis is the process in which a
cell divides itself into two halves,
each with an identical set of
chromosomes (Fig. 8.2).
The central regulator of this process
is the mitotic checkpoint, also known
as the spindle assembly checkpoint
or SAC, a signaling mechanism that
arrests the progression of metaphase
to anaphase until all chromosomes
are attached to the mitotic spindles.
This signal is akin to an
“anaphase wait” signal that is
generated at the kinetochores of
unattached chromosomes and is
extinguished once all
kinetochores are properly
attached to the spindles
(Fig. 8.2).
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 mis-segregation.
The proteins that control mitotic
checkpoint were originally
identified by screens for
mutations that bypassed the
ability of wild type S. cerevisiae
to arrest in mitosis in the
presence of spindle poisons.
The genes identified include
MAD (mitotic-arrest deficient),
MAD1, MAD2, MAD3 (BUBR1 in
humans), and BUB1 (budding
uninhibited by benzimidazole 1).
It was later found that these genes
are conserved in all eukaryotes.
When activated, these SAC proteins
target CDC20, which is a co-factor of
the ubiquitin ligase anaphase
promoting complex/cyclosome
(APC/C).

35. The “noncanonical” functions of cyclins,
CDKs and CKIs involve myriads of other
cellular processes such as transcription,
DNA damage repair, apoptosis, cell
differentiation, epigenetic regulation,
stem cell self-renewal, metabolism, and
the immune response.
Some of these functions are
performed by cyclins or CDKs
independent of their respective cell
cycle partners, suggesting that
there is substantial divergence in
their functions during evolution.
For example, D-type cyclins,
independent of any associated
kinase activity, are known to have
direct roles in regulating
transcription by interacting with
many transcription factors to
activate or repress transcription of
specific genes.
Similarly CDK6, but not CDK4, can
regulate angiogenesis and myloid
differentiation, respectively, by
modulating the transcriptional
activity of JUN and RUNX1.
In addition to regulating the cell
cycle, the trio of cyclins, CDKs, and
CKIs exerts important functions in
the repair of DNA damage
sustained from DSBs.
DNA DSBs are repaired by two
different mechanisms: homologous
recombination and NHEJ.269 Cyclin
D1 has been shown to localize to
DNA DSBs and to recruit RAD51,
which activates HR-mediated DNA
repair.
CDK2 was also shown to support
HR by promoting the interaction
between breast cancer type 1
susceptibility protein (BRCA1) and
the MRE11 exonuclease, leading to
the resection of DSBs.

36. PATHOLOGICAL
CONSEQUENCES OF CELL
CYCLE DEREGULATION
OR DYSREGULATION

37. Because regulation of the cell cycle is
central to the control of cell
proliferation, it is not surprising that
cancers are often the results of
deregulation or dysregulation of the
cell cycle.
Take colorectal cancer, for example,
recent genomicscale sequencing
studies have identified numerous
somatic mutations in genes that
possibly are involved in the formation
of cancer.
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 phosphatidylinositol 3-kinase,
catalytic, alpha subunit (PI3KCA).

38. Retinoblastoma (RB) Tumor
Suppressor Gene The RB
tumor suppressor protein
limits cell proliferation by
preventing entry into the S
phase of the cell cycle.
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.30
The INK4 family of CKIs,
particularly p16INKa,
directly inhibits activities of
the cyclin D-dependent
kinases, CDK4 and CDK6,
thus maintaining RB in its
active, antiproliferative
state.
Functional disruption of the
tumor suppressors, p16INKa
and RB, or overexpression of
the proto-oncogene
products, cyclin D1 and
CDK4, occur in many human
cancers, prompting the
speculation that disabling
the “RB pathway” is an
essential part of cancer
formation.

39. The tumor suppressor p53 is
mutated in more than 50% of
human cancers.303
It has been estimated that
cancers derived from over 50
human cell types or tissues
contain mutations in the p53
gene.
p53 is a labile protein but
accumulates in response to
cellular stresses from DNA
damage, hypoxia, or oncogenic
activation.
Upon stabilization and
activation, p53 initiates a
transcriptional program that
triggers either cell cycle arrest or
apoptosis.
Among the p53- responsive
genes are p21Cip1, BCL2-
associated X protein (BAX), and
mouse double-minute 2,
homolog (MDM2).321
While p21Cip1 regulates
progression of the cell cycle by
inhibiting cyclins (E, A, and B)-
CDK2 complexes, BAX causes
apoptosis.
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.
MDM2, in turn, is negatively
regulated by the ARF (alternative
reading frame) tumor suppressor
(p14ARF in humans and p19ARF
in mice).

40. Human FBXW7 exists in
three different isoforms,
α, β, and γ, each with a
unique amino terminal
end fused to a common
carboxyl terminal.361
The interaction between
FBXW7 and its substrates
depends on
phosphorylation of the
substrate within a motif
called the CDC4-
phophodegredron or
CPD.362
This feature enables
FBXW7 to simultaneously
regulate a host of
substrates by
ubiquitination.
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.
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. 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 dysregulated in many
human cancers, resulting
in dysfunction of the cell
cycle.
The consequences of cyclin E
deregulation are multitude
and include genetic instability,
centrosome amplification, and
fork collapse during DNA
replication.
Several studies
subsequently identified
cyclin E as a substrate for
FBXW7, which mediates
the phosphorylation- and
ubiquitination-dependent
degradation of cyclin E.
Thus, it appears that
tumorigenesis secondary
to cyclin E deregulation is
linked to altered function
of FBXW7/hCDC

41. These include chromosome dynamics (e.g., chromosome condensation, segregation, cohesion, and kinetochore-spindle interaction),
centrosome duplication, cell cycle checkpoints (include G1, S, G2, and the SACs), DNA damage repair pathway, and telomere functions.
Although there has not been a unified mechanism responsible for CIN, defects in several cellular processes have been causally linked to
its formation.
It has been suggested that CIN is the driving force for the formation of aneuploidy and tumorigenesis.
The remainders of the sporadic CRC have CIN, which are frequently aneuploid, that is, they exhibit alterations in the number of
chromosomes.
MIN tumors have mutations in the DNA mismatch repair (MMR) genes and accounts for approximately 15%–20% of sporadic CRC.
In colorectal cancer (CRC), there are two major forms of genetic instability: microsatellite instability (MIN) and chromosomal instability
(CIN).
Genetic instability has long been recognized as an integral part of human cancers.385

42. 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 lacking 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 centromeres protein E (CENP-E)
all have increased level of aneuploidy and CIN, with
the eventual formation of tumors in some animals.
Importantly, somatic mutations of many of the
same genes have been identified in human
cancers,415–423 indicating the importance of
the mitotic checkpoint in maintaining genomic
integrity

43. Wnt signaling is
normally absent
in a quiescent,
noncycling cell.
This is
accompanied by
the sequestration
of β-catenin in a
“destruction
complex” in the
cytoplasm that
includes APC,
Axin, casein
kinase 1 (CK1),
and glycogen
synthase kinase 3
(GSK3)
(Fig. 8.8A).451
This complex
leads to the
phosphorylation
of β-catenin,
which is then
degraded by
ubiquitin-
mediated
proteasomal
degradation.452–
459
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,
resulting in
accumulation of
free β-catenin in
the cytoplasm
(Fig. 8.8B).
Some of this free
β-catenin is
shuttled into the
nucleus where it
becomes
associated with
the transcription
factor, T-cell
factor (TCF) to
activate target
gene expression
(Fig. 8.8B).
Among the genes
stimulated by the
β-catenin/TCF
complexes are
those encoding
cyclin D1 and c-
Myc,
both of which are
critical for the
progression of
the cell cycle

45. Cell Cycle Regulators as Targets for
Cancer Treatment

46. • Because deregulation or dysregulation of the cell cycle is
frequently found in cancer, the cell cycle regulatory proteins
are logical targets for development of novel theories for
cancer.
• Recent studies have demonstrated that orally available
small-molecule inhibitors of the cyclin D-dependent CDK4
and CDK6, when combined with established therapies, have
potential in the treatment of certain cancers.
• A recently completed phase 3 clinical trial is an example of
a success story that the combination of palbociclib, a CDK4-
CDK6 inhibitor, and letrozole, an aromatase inhibitor, is highly
effective in the treatment of advanced breast cancer.

47. Quantitative analysis of cell cycle phase
durations

49. Figure 1. Phase-dependent
biosensor localization and
combinatorial usage. (A) The G1
phase biosensor construct
consists of the HDHB C-terminus
and tdimer2 (a dimeric red
fluorescent protein).
In G1 phase (post nuclear
envelope formation in the
daughter cells) the biosensor is
nuclear, as shown in the
schematic and transfected HeLa
cell (left).
As the cell progresses through
the cell cycle, the G1 phase
biosensor translocates to the
cytoplasm (G1/S), becoming
nuclear excluded in S and G2
phases.
Breakdown of the nuclear envelope in M
phase allows for fluorescence to spread
throughout the rounded cell, with
exclusion only at the condensed
chromatin (right). Time is in
hours:minutes. (B) The nuclear localized S
phase biosensor consists of an NLS (SV40
nuclear location signal), EYFP, a linker (18
hydrophilic amino acids) and PCNA.
Epifluorescence timelapse
images of a HeLa cell (top) and a
schematic (bottom) show S
phase biosensor localization
through one cell cycle.
The nuclei of the schematic S phase cell have been
enlarged to better illustrate puncta formation and
their change in morphology, which indicates
replicative progression. Time is in hours:minutes.
(C) Confocal images of a HeLa cell with a punctate S
phase biosensor (green) and cytoplasmic G1 phase
biosensor (red) denotes S phase DNA replication.
(D) Single frame analysis of coexpressed G1 and S
phase biosensors allows for the identification of the
four phases.
When the G1 phase biosensor is nuclear and the S phase
biosensor is nuclear but not punctuate, the cell is in G1
phase (arrows). A cytoplasmically localized G1 phase
biosensor and a punctate S phase biosensor identify S
phase cells (*). G2 cells have cytoplasmic G1 phase
biosensor fluorescence and non-punctate S phase
biosensor fluorescence (circle).
Coalignment of the G1 and S phase
fluorescence in the rounded mitotic
cell body occurs post nuclear
envelope breakdown (square). S
phase puncta are not always easy to
identify, especially in early and mid S
phase (