chapter 6 - cell cycle

8/13/2019 Chapter 6 - Cell Cycle

http://slidepdf.com/reader/full/chapter-6-cell-cycle 1/26

Chapter 6: Cell Cycle

Cell Cycle: Introduction

Cell division is a process that must be carried out with absolute fidelity. The program of

generating an adult organism from a single zygote involves countless cell duplications, each

requiring the precise partitioning of genetic material and most other cellular components to

daughter cells. The division process then continues during adult life to replenish essential

cells restricted to a limited lifespan. As a result, organisms have evolved cell-duplication

strategies that include redundant safeguards to prevent errors or, if errors occur, to correct

them. Nevertheless, errors do occur at a measurable frequency, and mutations accumulated

over time can weaken protective mechanisms, rendering the genome increasingly vulnerable

to challenges. The resulting loss of genetic and genomic stability has serious implications for

survival in that it is a major contributing factor to the development of malignancy. Indeed,

cancer is one of the leading causes of mortality in humans.

In this chapter, the basic principles of mammalian cell division and the mechanisms that

have evolved to safeguard the integrity of the process are reviewed. Then there is a

discussion of how the normal control mechanisms of cell division and protective safeguards

become subverted in cancer cells. It is hoped that, ultimately, detailed knowledge of cell

division in normal and cancer cells will lead to rational effective therapeutic approaches.

ell-Cycle Engine

Although the details of cell division vary considerably across phylogenetic lines and even in

different cell types within the same organism, the underlying infrastructure that mediates

and controls the cell division process is remarkably conserved. If one compares yeast cells

and mammalian cells in culture, perhaps the two most aggressively studied model cell

division systems are not only the respective cell division cycles organized along a similar

scheme, but many of the proteins used in the cell division pathway are easily recognizable as

being evolutionarily related. Indeed, some of these proteins are so highly conserved that

they are functional in the heterologous organism despite a billion years of divergent

evolution.

Phases of the Cell Cycle

As alluded to previously, the basic organization of the cell cycle is highly conserved in

eukaryotic evolution. In 1951, Howard and Pelc,1 studying the division of plant root cells,

separated the process into four phases eventually referred to as GAP1, synthetic

phase, GAP2, and mitosis. The shorthand that emerged from this descriptive work (G1, S

phase, G2, and M phase or mitosis) has been the lens through which all subsequent dividing

cells have been observed, and the four successive phases are referred to collectively as

the cell cycle. The key observation made by these investigators was that the events that

together make up the cell division process do not all occur continuously. Specifically,

although growth and protein synthesis occur constantly for the most part, synthesis of DNA

occurs only during a discrete interval. The preceding phase was designated GAP1 or G1, and

http://lwwoncology.com/Textbook/Content.aspx?aid=8750961#8750961






the subsequent phase before cell division was referred to as GAP2 or G2. Although at the

time little could be said concerning what a cell did during these silent ―gap‖ phases, it is now

known that these are not idle periods in a cell’s life but the intervals in which most regulation

of the cell cycle is specifically exerted. A large amount of information, originating from the

external environment and the cell’s internal milieu, is integrated during the G1 andG2 intervals and used to determine whether and when to proceed into S phase and M phase,

respectively.

Mitosis, the most visibly dynamic interval of the cell cycle, has itself been traditionally

subdivided into five phases: prophase, prometaphase, metaphase, anaphase, and telophase.

In metazoans and plants (as opposed to fungi) mitosis entails a particularly dramatic change

of state for the cell. During prophase most of the internal membranous compartments of the

cell, including the nucleus, are disassembled and dispersed. Replicated chromosomes

(chromatids) are condensed into paired compact rods, and a bipolar microtubule spindle is

assembled. Biosynthesis of proteins (transcription and translation) largely ceases. Duringprometaphase, chromosomes form bivalent attachments to the spindle, driving them to the

cellular equator. Proper alignment of paired chromatids on the spindle is indicative of

metaphase. During anaphase, the paired sister chromatids lose cohesion and microtubule

forces separate the chromatids and pull them to opposite poles of the cell. During telophase,

the events of prophase are reversed: The nuclei and other membrane structures reassemble,

the chromosomes decondense, and protein synthesis resumes. After mitosis, the two

daughter cells pull apart and separate in a process known ascytokinesis.

Current knowledge of the cell cycle has accrued historically from a number of different

experimental approaches and systems. In the early 1970s, experiments carried out by fusingmammalian cells in different cell-cycle phases revealed the existence of dominant inductive

activities for the S phase and the M phase.2,3 Shortly thereafter, similar inductive activities

were isolated from mature frog eggs arrested at meiotic metaphase II and shown to be

capable of inducing G2 oocytes to enter into meiotic divisions,4 equivalent in many respects

to mitosis. At the same time, genetic analysis of cell division in yeast revealed that the

products of individual genes controlled specific events in the cell cycle and that these events

could be organized in pathways, much like metabolic pathways.5 Eventually, all of these

lines of investigation converged in the 1980s, leading to the discovery of cyclin-dependent

kinases (CDKs).

Cyclin-Dependent Kinases

Arguably the most significant advance in understanding cell-cycle regulation was the

discovery of CDKs.6These are binary, proline-directed, serine-threonine–specific protein

kinases that consist of a catalytic subunit (the CDK) that has little if any intrinsic enzymatic

activity and a requisite positive regulatory subunit known as a cyclin. In yeast, one CDK and

numerous cyclins carry out cell-cycle regulatory functions, whereas in mammals, these same

functions are carried out by a number of different CDKs and cyclins. In yeast, in which

multiple cyclins activate the same CDK (CDK1, also known as Cdc28 or Cdc2, depending on

the species) for distinct cell-cycle tasks, it is clear that most if not all substrate specificity

beyond a rather degenerate primary structure target consensus lies in the cyclin subunit. In






















mammals, it is likely that substrate specificity is shared by CDK and cyclin subunits.

Although not all pairwise combinations are permitted, there are enough combinatorial

possibilities to create a significant level of substrate specificity.

CDKs have a structure similar to that of other protein kinases, consisting of two globular

domains (the N-lobe and C-lobe) held together by a semiflexible hinge region. Protein

substrates bound by the active enzyme are thought to fit into a cleft between the two

domains. The N-lobe contains the adenosine triphosphate (ATP)–binding site. Studies

comparing CDKs and CDK-cyclin complexes based on x-ray diffraction crystallography

indicate that the primary role of the cyclin, in addition to substrate docking functions, is to

realign critical active site residues into a catalytically permissive configuration and to open

the catalytic cleft to accommodate substrates.7 Once bound to a cyclin, the CDK active site is

configured similarly to other protein kinases that do not require cyclin binding.

The known CDKs and cyclins and their presumptive intervals of function in the mammalian

cell cycle are summarized in Figure 6.1. For the most part, the functional intervals of CDKs

are determined by the accumulation and disappearance of cyclins. Whereas CDKs tend to be

expressed at a constant level through the cell cycle, cyclin accumulation is usually dynamic,

regulated at the level of biosynthesis and degradation (discussed in greater detail in ―Cell-

Cycle Machinery‖ ). To summarize, three partially redundant D-type cyclins (D1, D2, and D3)

activate two partially redundant CDKs (CDK4 and CDK6). Although, unlike most other

cyclins, D-type cyclins do not appear to be expressed with high periodicity in cycling cells,

the interval at which their primary activating function is thought to occur is from mid-to-late

G1 to direct phosphorylation of the cell-cycle inhibitor pRb and related proteins p107 and

p130. Phosphorylation of these proteins by cyclin D–CDK4/6 inactivates their negativeregulatory functions, allowing progression into S phase.8,9

Figure 6.1. Windows of cyclin-dependent kinase (CDK) function in the cell cycle.

D-type cyclins (cyclins D1, D2, and D3) activate CDK4 and CDK6 for functions extendingfrom middle G1 to the G1 /S-phase transition. E-type cyclins (cyclins E1 and E2) activate

CDK2 for functions at the G1 /S-phase boundary, probably extending into early S phase.

Cyclin A activates CDK2 for functions extending from the G1 /S-phase boundary and

extending into G2. Cyclin A is known to interact with CDK1 as well; however, no specific

function for this complex has been identified. Finally, cyclin B activates CDK1 at the G2 /M-

phase boundary with activity that lasts until cyclin B is degraded during anaphase.

Unlike D-type cyclins, E-type cyclins (E1 and E2) are expressed with high cell-cycle

periodicity, accumulating in late G1 and declining during S phase. E-type cyclins activate

CDK2, and the fact that premature expression of cyclin E1 leads to accelerated entry into S

phase10,11 has suggested that the target(s) must be proteins responsible for initiation of

DNA replication. However, the essentiality of cyclin E–CDK2 in this context has been put into




http://windowreference%28%27reference%27%2C%27/Popup/index.aspx?aID=8750887%27);












http://windowreference%28%27reference%27%2C%27/Popup/Index.aspx?aID=8750887%27);



















question by the demonstration that cells from cyclin E1/E2 nullizygous mouse embryos can

cycle with reasonably normal kinetics and can certainly initiate DNA replication.12 The most

likely explanation for the dispensability of E-type cyclins for S phase functions is redundancy

with cyclin A, which also activates CDK2.

Cyclin A accumulates initially at the G1 /S-phase boundary and persists until prometaphase of

mitosis. It has been best characterized as an activator of CDK2; however, it has also been

reported to form complexes with CDK1. It is presumed that CDK2, activated by E-type

cyclins and cyclin A, promotes cell-cycle progression from the G1 /S boundary through the

G2 interval.

At this time, B-type cyclins, in conjunction with CDK1, are responsible for getting cells into

and through mitosis. Although mammalian cells express a number of B-type cyclins, only

cyclin B1 appears to be essential. Cyclin B1 accumulates through S phase and G2 and then is

degraded at the metaphase- anaphase transition. It should be pointed out that the CDK

family is extensive and that eukaryotes possess many additional CDKs that ostensibly have

nothing to do with cell-cycle regulation.

The blueprint for CDK function through the mammalian cell cycle presented here is based on

a large body of experimental evidence and most likely accounts for primary activities at each

cell-cycle stage. However, this model does not account for potential redundancy of CDK

function. Recently, however, it has been demonstrated that a CDK2-nullizygous mouse is

viable, and furthermore, relatively normal.13,14 Investigation of embryonic fibroblasts from

these mice has suggested that in the absence of CDK2, CDK1 can carry out the functions

normally attributed to CDK2.15 However, the contribution of CDK1 to these functions in

unperturbed cells remains to be determined.

Modes of Cyclin-Dependent Kinase Regulation

Because the activity of CDKs is central to cell survival, these enzymes are, of necessity,

highly regulated.6 As a result, a number of diverse regulatory mechanisms have evolved to

allow for integration of environmental and internal signals (Fig. 6.2). A primary mode of CDK

regulation is the availability of activating cyclins, as alluded to previously. For most cell-cycle

regulatory CDKs, the relevant cyclins exhibit a distinct temporal program of accumulation

and degradation, determining a precise window of CDK activation. Although D-type cyclins

tend not to be highly regulated in cycling cells, they are strongly down-regulated as cells exit

the cell cycle into a nonproliferative state and then resynthesized in response to mitogen

stimulation and cell-cycle re-entry. The genes encoding cyclin E1 and E2 are transcribed

periodically late in G1 and up to the G1-S phase transition. This, coupled with ubiquitin-

mediated proteolysis of cyclin E in active cyclin E–CDK2 complexes, creates the observed

window of cyclin E accumulation from late G1 to mid-S phase.


























Figure 6.2. Principles of cyclin- dependent kinase (CDK) regulation.

Because CDK catalytic subunits are inactive when unbound to cyclins, the first level of

regulation is through the expression and availability of cyclins. The second level of regulation

appears to constitute a housekeeping function in that cyclin binding stimulates an essential

phosphorylation of a threonine within a structural feature known as the T loop. Binary

complexes with phosphorylated T loops are active. Such active kinase complexes can be

subjected to negative regulation in two ways. Additional phosphorylation events on threonine

14 and tyrosine 15 (or the equivalent residues) render the kinase inactive. In addition, theformation of ternary complexes with CDK inhibitory proteins (Ckis) promotes an inactive

state. One class of Cki, INK4, specific for CDK4 and CDK6, can inhibit by forming binary

complexes directly with the CDK.

Like cyclin E, the accumulation of cyclin A is determined by periodic transcription. However,

unlike cyclin E, cyclin A remains stable in active CDK2 complexes. The timing of ubiquitin-

mediated proteolysis of cyclin A is determined by activation of a protein-ubiquitin ligase

known as the anaphase-promoting complex/cyclosome (APC/C) in prometaphase. Thus, the

window of cyclin A accumulation is from the G1-S transition until early in mitosis. Finally, B-

type cyclin accumulation is also linked to periodic transcription. In this case, transcription

begins in late S phase and persists through G2. Similarly to cyclin A, B-type cyclins are

targeted for ubiquitin-mediated proteolysis by the APC/C during mitosis, although their

disappearance occurs slightly later in mitosis than that of cyclin A.

It is interesting to note that periodic transcription of cyclins E, A, and B mRNAs relies

primarily on negative regulation. For cyclin E, an element known as CERM (cyclin E repressor

module) binds a repressor complex containing the repressive member of the E2F family of

transcription factors, E2F4, as well as the Rb-related protein p107 and a histone

deacetylase.16 Inactivation of the repressive complex in late G1 via phosphorylation of p107

by CDK4/6 allows the constitutive transcription factor, Sp1, to drive cyclin E transcription.

Transcription of cyclin A and B mRNAs is similarly regulated. In this case, the repressor

element is known as cell cycle genes homology region (CHR)17 but the corresponding

repressor complex has not yet been well characterized. However, once repression is relieved,

transcription of both cyclin A and B mRNAs is driven the constitutive transcription factor, NF-

Y.

A second important mode of CDK regulation is by phosphorylation. CDKs require an

activating phosphorylation on a structural feature designated the T loop. Phosphorylation

induces a movement of the T loop that has global effects on CDK structure, including an

increase in CDK-cyclin contacts and changes in the substrate-binding site.18 In most if not

all instances, T-loop phosphorylation appears to constitute a housekeeping function that




















occurs concomitant with cyclin binding. However, negative regulatory phosphorylation of

CDKs is a highly dynamic process. Proper cell-cycle regulation of CDK1, in particular,

requires phosphorylation on two residues within the N-lobe, adjacent to the ATP-binding site:

threonine 14 and tyrosine 15. During the normal course of the cell cycle, as cyclin B–CDK1

complexes accumulate, they are immediately phosphorylated at these sites and thereby keptinactive. This allows stockpiling of the large numbers of cyclin B–CDK1 complexes required

for efficient entry into mitosis and maintaining them in an inactive state during late S phase

and G2. At the G2 /M-phase boundary, there is concerted dephosphorylation of these residues,

causing cells to advance rapidly into mitosis. Although CDK2 and CDK4 have also been

reported to undergo negative regulatory phosphorylation at the homologous residues, the

function(s) of this regulation are not as clear-cut (but see ―DNA Damage Checkpoints‖ ).

A third mode of CDK regulation is through the action of inhibitory proteins that can form

either binary complexes with CDKs or ternary complexes with cyclin-CDK dimers. These exist

in three major families. The INK4 family consists of four members (p15, p16, p18, and p19).All are composed of a series of conserved structural motifs known as ankyrin repeats and

they specifically target CDK4 and CDK6. The mechanism of action of these inhibitors is to

bind the CDK subunit and, by causing a rotation of the N-lobe relative to the C-lobe,

constraining the kinase in an inactive conformation and, in addition, precluding cyclin

binding.19 The Cip/Kip family consists of three members in mammals: p21Cip1, p27Kip1, and

p57Kip2. All contain a conserved amino terminal cyclin-CDK–binding inhibitory domain and a

divergent C-terminal domain possessing other less well-characterized functions. Although

these have been characterized primarily as potent inhibitors of CDK2 and have more recently

been shown to also be effective CDK1 inhibitors, the case for inhibition of CDK4 and CDK6 is

less certain. Whereas Cip/Kip inhibitors are clearly capable of inhibiting CDK4 and CDK6 at

high concentration, it is not clear that these conditions are met in vivo, and the situation is

further complicated by the finding that Cip/Kip inhibitor binding is actually required to

provide a chaperonin or assembly function for the efficient formation of active cyclin D–CDK4

complexes.20 In the case of cyclin A–CDK2, where structural studies have been carried out,

it appears that the Cip/Kip inhibitors first anchor via a high-affinity interaction with the

cyclin.21 This then allows the inhibitor polypeptide to invade and deform the N-lobe, thus

interfering with ATP binding and catalysis.16 The final class of inhibitors consists of two

members of the pRb protein family, p107 and p130. Although these proteins have well-

characterized functions as transcriptional inhibitors, they also are potent cyclin E/A–CDK2inhibitors. p107 and p130 each contain cyclin-binding and CDK-binding sites that collaborate

to confer inhibitory activity.

A final mode of CDK regulation is via control of nuclear import/export. This level of regulation

is most obvious for cyclin B–CDK1 complexes, which are kept out of the nucleus via active

nuclear export until late G2, when phosphorylation inactivates cis-acting nuclear export

signals allowing nuclear accumulation.22 Sequestration of cyclin B–CDK1 in the cytoplasm is

a redundant mechanism, along with negative regulatory phosphorylation of CDK1, for

preventing premature phosphorylation of mitotic targets.



























Induction of Cell-Cycle Phase Transitions

The cell cycle is composed of two action phases, S phase and M phase, in which the genetic

material is duplicated and the components of a mother cell are divided into two daughter

cells, respectively. The intervening phases, G1 and G2, are thought to exist primarily to allow

time for cell growth and for regulatory inputs. Therefore, from the point of view of regulatory

theory, cell proliferation is controlled operationally at two key transitions: that between

G1 and S phase and that between G2 and M phase. The important characteristic of these two

transitions is that, once initiated based on integration of regulatory signals, they must be

executed decisively to maintain genetic and genomic integrity. This is accomplished by using

a combination of positive and negative modulators to set up the equivalent of a molecular

capacitor.

In cycling mammalian cells, the programmed accumulation of cyclins E and A via

transcriptional induction provides the positive impetus for the G1-S phase transition.

However, these kinases are kept in check by the action of Cip/Kip family inhibitors. If the

internal and external environments are permissive for proliferation, the continued

accumulation of cyclins will eventually titrate the inhibitors, allowing the latter to be

phosphorylated by free cyclin-CDK complexes. Phosphorylation then marks these inhibitors

as targets of ubiquitin-mediated proteolysis. The concerted destruction of CDK inhibitors and

concomitant activation of the entire pool of CDK complexes assure that the transition into S

phase is rapid and irreversible.

Although the details of its regulation are somewhat different, the strategy underlying control

of the G2-M transition is similar. Cyclin B–CDK1 complexes accumulate starting near the end

of S phase but are held in check not by CDK inhibitors but by negative regulatory

phosphorylation of CDK1. This phosphorylation on threonine 14 and tyrosine 15 is carried out

by kinases Wee1 and Myt1. Entry into M phase is signaled by the rapid dephosphorylation of

T14 and Y15, resulting in activation of CDK1. This dephosphorylation is carried out by

specialized protein phosphatases, CDC25B and CDC25C. The concerted dephosphorylation of

CDK1 depends on activation of CDC25 isoforms by phosphorylation, as well as ubiquitin-

mediated proteolysis of Wee1, also in response to phosphorylation. Although the initial

activation of CDC25 isoforms is thought to be carried out by other protein kinases, such as

Plk1, CDC25B and C are also activated by cyclin B–CDK1, establishing a positive feedback

loop. These positive feedback dynamics leading to the simultaneous activation of a largeaccumulated pool of cyclin B–CDK1 assures that entry into mitosis is decisive. The turnover

of Wee1 enforces irreversibility. Because entry into mitosis involves dismantling many of the

cell’s components and organelles, as well as construction and use of a complex apparatus for

segregating the cell’s genetic material, mitosis is a period of particular vulnerability, and

therefore it is important that this transition and subsequent events be carried out rapidly and

efficiently.

An important secondary transition that occurs within M phase is that between metaphase

and anaphase (Fig. 6.3).23 To preserve genomic integrity, all duplicated chromosomes must

be aligned along the cell’s equator and properly attached to microtubules of the mitoticspindle. The trigger for separation of sister chromatids and their movement to opposite poles











E.27–29 The second family of protein-ubiquitin ligases that is critical for cell-cycle control is

known collectively as the APC/C . The APC/C is a large complex consisting of 12 core subunits

and 1 of 2 specificity factors, CDC20 and CDH1. Unlike SCF ubiquitin ligases, targeting of

substrates by the APC/C is determined by ligase activation rather than substrate activation.

APC

CDC20

is active from metaphase until the end of mitosis as a result of periodic accumulationand degradation of CDC20 itself. APCCDH1, on the other hand, which is negatively regulated by

CDK-mediated phosphorylation, is activated on mitotic exit and during the subsequent

G1 interval when CDKs are inactive. In this manner, important mitotic targets, such as cyclin

A, cyclin B, and securin (as well as many others), are degraded during mitosis and prevented

from reaccumulating during the subsequent G1 interval.

Regulation of the Cell Cycle

To preserve organismic function and integrity, the cell cycle must be regulated at a number

of levels. These include entry into and exit from proliferation mode, coordination of cell-cycle

events, and specialized responses that increase the probability of surviving a variety of

environmental and internally generated insults.

Quiescence and Differentiation

The most fundamental aspect of cell-cycle control is the regulation of entry and exit. For

mammalian cells, the decision to enter or exit the proliferative mode is based on

environmental signals such as mitogens, growth factors, hormones, and cell-cell contact, as

well as on internal differentiation programs. If the state of cell-cycle exit is reversible, it is

referred to as quiescence. If it is in the context of terminal differentiation, cell-cycle exit may

merely be one component of a differentiation program. Although cells entering quiescence

and postmitotic differentiation vary from each other in many respects, from the perspective

of cell-cycle control, they have much in common. First, cell-cycle exit is usually associated,

at least initially, with an accumulation of G1 /S CDK inhibitors. Members of the INK4 family,

targeting CDK4 and CDK6 and members of the Cip/Kip family, as well as the Rb-related

protein p130, all targeting CDK2, are up-regulated. This causes accumulation of cells in G1,

from where cell-cycle exit can occur. Next, or simultaneously, the positive cell-cycle

machinery is dismantled by down-regulation of CDKs and cyclins, primarily at the

transcriptional level. In the case of quiescence, cell-cycle exit is paralleled by a reduced rate

of protein synthesis, indicative that cells have entered a resting state.

Entry into and exit from quiescence are mediated largely by growth factors and mitogens

that interact with cell surface receptors. These in turn are linked to intracellular signaling

cascades that up-regulate the rate of protein synthesis as well as the transcription of genes

that promote proliferation, such as those encoding CDKs and cyclins. The two best-

characterized signaling pathways in this context are the mitogen-activated protein

kinase/extracellular signaling–regulated kinase pathway30 and the phosphoinositide 3 (PI3)

kinase/AKT pathway,31 shown in Figure 6.4. Whereas the mitogen-activated protein

kinase/extracellular signaling–regulated kinase pathway tends to stimulate expression of

genes required for proliferation, the PI3-kinase/AKT pathway primarily stimulates protein

synthesis and growth but also affects key cell-cycle regulatory proteins. Just as the presence

of growth factors and mitogens stimulates these pathways, promoting cell-cycle entry, their






















removal shuts down these pathways, promoting quiescence. This is the basis for the

reversibility of the quiescent state.

Figure 6.4. Growth factor (GF)/mitogen stimulator in response to occupancy of

many GF receptors (GFRs) by ligand depends on the small guanosine

triphosphatase transducer Ras.

Receptor activation leads to phosphorylation of the receptor cytoplasmic domain. The

phosphorylated receptor assembles a complex that includes Ras and its activated nucleotide

exchange factor, son of sevenless (SOS), leading to activation of Ras. Activated Ras can then

stimulate two important signal transduction pathways: the extracellular signaling–regulated

kinase (ERK) pathway and the phosphoinositide 3 kinase (PI3K) pathway. Activated Ras

stimulates the protein kinase activity of Raf, activating a protein kinase cascade consisting of

Raf, MEK, and ERK. Activated ERK then translocates into the nucleus, where it

phosphorylates and activates transcription factors, notably Elk-1. Genes important for growth

and division are then transcribed. Activated Ras also stimulates PI3K activity, leading to the

accumulation of phosphatidylinositol 3,4,5-triphosphate. This in turn stimulates the proteinkinase activity of phosphoinositide-dependent kinase 1 (PDK1), activating a protein kinase

cascade consisting of PDK1, AKT, and mTOR. Activation of this signal transduction pathway

has the effect of stimulating translation and growth. AKT phosphorylates and inhibits the

protein kinase glycogen synthase kinase 3 (GSK3 β), thereby activating EIF2B required for

translational initiation. mTOR phosphorylates and inhibits the protein phosphatase PP2A,

thereby activating EIF4E, also required for translational initiation. Finally, mTOR

phosphorylates and activates pp70S6 kinase, which in turn phosphorylates and activates

ribosomal subunit S6.

Antimitogenic Signals

An important aspect of control of cell division in mammals is antimitogenic signaling. Just as

mitogens and growth factors bind to transmembrane receptors and use signal transduction

pathways and downstream transcriptional programs to stimulate proliferation, parallel

systems antagonize proliferation. The classic example of an antimitogenic signal is the effect

of transforming growth factor- β(TGF- β) on epithelial cells (Fig. 6.5).32 TGF- β, a cytokine,

binds to a specific heterodimeric transmembrane receptor that, when occupied by ligand,

phosphorylates a class of transcription factors, known as SMADs. These phosphorylated

SMADs heterodimerize with non−receptor-interactive SMADs and translocate to the nucleus,

where they complex with DNA-binding transcription factors and coactivators to transactivate

specific genes. Relevant to cell-cycle regulation, stimulation of the TGF- βsignaling pathway

promotes transcription of the gene encoding p15. p15 is an INK4 class CDK inhibitor that



















specifically inactivates CDK4 and CDK6. However, the effects of p15 accumulation on cell-

cycle regulators are more global than inhibition of CDK4/6.33 INK4 inhibitors such as p15

have a secondary effect of displacing a pool of the Cip/Kip inhibitor p27 from cyclin D–

CDK4/6 complexes, allowing it to then target and inactivate cyclin E–CDK2 and cyclin A–

CDK2 (Fig. 6.5). Thus, exposure of epithelial cells to TGF- β has the effect of inhibiting G1 andS phase CDK activities, thereby causing G1 arrest.

Figure 6.5. Transforming growth factor- β (TGF- β) antimitogenic pathway.

Occupancy of the heterodimeric TGF- β receptor by ligand leads to phosphorylation of a class

of transcription factor known collectively as R-SMADs. Phosphorylated R-SMADs then

dimerize with nontargeted cofactors known as CoSMADs and translocate to the nucleus.

There the R-SMAD/CoSMAD dimers complex with DNA-binding transcription factors and

transcriptional coactivators to stimulate transcription of specific genes. One of the key

targets of TGF- β signaling is the gene encoding p15/INK4b, a CDK4/6 inhibitor. p15, by

binding to CDK4 and CDK6, inhibits these kinases and displaces a large pool of CDK4/6-bound p27, which is then free to inhibit CDK2 complexes. The result is G1 arrest. ATP,

adenosine triphosphate; TF, transcription factor; CoAct, coactivator.

Interestingly, in many cancers of epithelial origin, the response to TGF- β has been

abrogated, suggesting that this and similar response pathways have an important role in

maintaining control of proliferation. Interferons comprise another class of cytokines that

have antiproliferative effects on many cell types. Although the receptors used and signaling

pathways are distinct from those used by TGF- β, the ultimate effects on the cell-cycle

machinery are similar: up-regulation of CDK inhibitors and down-regulation of cyclins.

CheckpointsCells are constantly faced with insults, resulting in damage that can threaten their survival.

These insults can be generated internally as chemically active by-products of metabolism or

can originate in the external environment; for example, chemical agents or radiation. As a

result, mechanisms have evolved to remove damaged molecules and make necessary

repairs. In instances in which cell-cycle progression would be harmful or catastrophic before

repair of damage, further mechanisms have evolved to delay progression pending repair.

These are called cell-cycle checkpoints.34 The necessity of checkpoints can be easily

envisioned for genotoxic agents. Cells are particularly susceptible to the harmful effects of

DNA damage at two points in the cell cycle: S phase and M phase. Unrepaired DNA damage

poses a number of problems for cells undergoing DNA replication. Chromosomal lesions























present physical barriers to replication forks. Replication that does traverse regions of

unrepaired DNA damage is likely to be error-prone, resulting in accumulation of mutations.

Likewise, segregation of severely damaged chromosomes at mitosis might lead to loss of

genetic information, seriously threatening the survival or integrity of daughter cells.

Therefore, cells possess mechanisms for preventing DNA replication and mitosis in responseto genotoxic stress. Although the scope of this review does not permit a detailed description

of all known checkpoints, those thought to be most basic to cell survival are characterized

here.

DNA Damage Checkpoints

Although DNA damage exists in many forms, ranging from chemical adducts to double-

strand breaks, they all pose similar problems for proliferating cells. As previously stated,

impeded and error-prone DNA replication and loss of genetic material during mitosis are

some of the likely consequences in the absence of DNA damage checkpoints. Therefore, cell-

cycle progression is blocked at three points: before S phase entry (the G1 DNA damagecheckpoint), during S phase (the intra-S phase DNA damage checkpoint), and before M

phase entry (the G2 DNA damage checkpoint). Although the responses to different types of

DNA damage are not identical, they are similar enough to generalize. DNA damage of various

forms is first detected by DNA-bound protein complexes that serve as sensors. In

mammalian cells, two related atypical protein kinases that share homology with lipid kinases,

ATM and ATR,35 are primary signal transducers that are activated by DNA damage at all

points in the cell cycle. A key effector of the G1 and G2 checkpoint responses is a

transcription factor known as p53.36,37 In response to DNA damage, p53 is activated and

stabilized leading to increased levels. The principal transcriptional target of p53 in the

context of the G1 checkpoint is the Cip/Kip inhibitor p21Cip1. The resulting high levels of p21

block CDK2 activity and possibly CDK4 and CDK6 activity, leading to G1 arrest. An additional

transcriptional target of p53, GADD45, inhibits CDK1, thereby contributing to the G2 DNA

damage checkpoint. Another p53-dependent mechanism contributing to checkpoint-mediated

G2 arrest is through transcriptional repression of the genes encoding cyclin B1 and CDK1.

This occurs via direct interaction p53 and NF-Y, the positive transcriptional activator of these

genes. However, although p53-dependent mechanisms are required for long-term

maintenance of arrest, the primary mechanism underlying the immediate G2 DNA damage

checkpoint is p53-independent. It involves one of two effector protein kinases known

as chk1 and chk2 that have the effect of inhibiting CDC25C,38 which carries out the

activating dephosphorylation of CDK1. Therefore, in response to DNA damage, G2 cells

accumulate inhibited cyclin B–CDK1 complexes and are incapable of entering into mitosis.

The intra-S phase DNA damage checkpoint response appears to be p53-independent but

requires the chk1 or chk2 kinases, or both. A key target is CDC25A, responsible for

activating CDK2 by dephosphorylation. In response to DNA damage, phosphorylation of

CDC25A by chk1 or chk2 leads to its destabilization via ubiquitin-mediated proteolysis and

thus the accumulation of inactive CDK2 complexes39 phosphorylated on threonine 14 and

tyrosine 15. Because ongoing DNA replication requires the activity of CDK2, DNA synthesis

ceases until damage is repaired.

Replication Checkpoint






















Under normal circumstances, DNA replication is complete well before the time when the

accumulation and activation of cyclin B–CDK1 would drive cells into mitosis. However,

through the action of toxins or the rare but finite probability that the duration of S phase will

be excessively long, situations can be encountered in which completion of replication extends

beyond the normal time of mitotic induction or replication is blocked entirely. Under suchcircumstances, it is necessary to delay or block entrance into M phase accordingly, as

segregation of incompletely replicated chromosomes would be catastrophic, leading to

chromosome breaks and/or nondisjunction events. Although the signaling pathways are

somewhat different, the replication checkpoint ultimately functions like the G2 DNA damage

checkpoint in that mitotic entry is blocked by inhibiting CDC25C via the action of chk1, thus

preventing activation of CDK1.

Spindle Integrity Checkpoint

The actual act of division is a dangerous time for a cell. It requires aligning duplicated

chromosomes by attaching them via bipolar attachments to the spindle and then separatingthe chromatids so that each daughter cell gets a full complement. Errors result in aneuploidy,

an extremely undesirable outcome. As a result, assembling a mitotic spindle and attaching

chromosomes to it are extensively monitored processes. The mechanism of delay at

prometaphase or metaphase in response to spindle defects or improper chromosome

attachment is referred to as the spindle integrity checkpoint .40 The sensor for this

checkpoint consists of a number of proteins that reside at the chromosome kinetochores,

sites of spindle microtubule attachment. The target is the essential APC/C cofactor, CDC20.

Unattached or improperly attached kinetochores not experiencing an appropriate level of

tension indicative of bipolar attachment inhibit CDC20 function. This in turn prevents the

ubiquitylation and degradation of the anaphase inhibitors, securin and cyclin B. As a result,

cells are prevented from initiating anaphase until all kinetochores are properly attached to a

bipolar spindle (Fig. 6.3).

Restriction Point

Cells deprived of an essential nutrient or growth factor are blocked from cell-cycle

progression at a point in mid-G1.41 Cells that have already passed this point, termed

the restriction point or R, enter into S phase and complete the current cell cycle before

arresting in the subsequent G1 interval. In contrast, G1cells that have not reached the

restriction point arrest immediately. The molecular basis for the restriction point has

remained elusive. Initially it was thought that passage through the restriction point was a

manifestation of G1 CDK activation and/or phosphorylation the pRb family of transcriptional

inhibitors. However, more recent work has indicated that CDK activation and pRb

phosphorylation occur after passage through the restriction point.42,43 Significantly, most

malignant cells do not have a functional restriction point, which presumably helps them

evade normal growth control signals.

Senescence

All normal mammalian cells have a finite proliferative lifespan. As cells approach the end of

their proliferative capacity, they enter a state referred to as replicativesenescence.44,45 Although the reasons for programmed senescence are not known, it has





























Figure 6.6. pRb pathway.

pRb is a critical negative cell-cycle regulator that links growth factor (GF) signaling pathways

to cell-cycle progression. One of the principal functions of pRb is to interact with E2F family

transcription factors and, by recruitment of corepressors, to maintain many genes encoding

proteins that are important for cell-cycle progression in a tightly repressed state. GF and

mitogen-signaling pathways relieve this repression by stimulating accumulation of D-type

cyclins on receptor occupancy. The resulting activation of CDK4 and CDK6 leads to

phosphorylation of pRb and concomitant inactivation of its repressive functions. p16 is a CDK

inhibitor of the INK4 family that down-regulates this pathway by inhibiting CDK4. It should

be noted that all elements marked by an asterisk are found mutated or deregulated, or both,

in human cancer. GFs, GF receptors (GFRs), and D-type cyclins are frequently overexpressed

or deregulated. p16 is often not expressed or is underexpressed. Mutant versions of CDK4

that cannot bind p16 have been identified in human cancers. Finally, the gene encoding pRb

is frequently mutated in cancer.

Cell Cycle and Cancer

Cancer is partly a disease of uncontrolled proliferation. Because the proliferation of cells

within an organism is normally tightly controlled by redundant regulatory pathways, it is not

surprising that cell-cycle and checkpoint genes are often found misregulated or mutated in

cancer. Genes in which mutations give rise to a gain of function or an enhanced level of

function, leading to malignancy, are referred to as protooncogenes. Protooncogenes usually

encode growth- or division-promoting proteins. Genes that give rise to loss of function

mutations that lead to malignancy are referred to as tumor suppressor genes. Tumor

suppressor genes usually encode negative regulators of growth and proliferation that protect

cells from malignancy. Some cell-cycle genes commonly mutated or misregulated in cancer

are listed in Table 6.1. Whereas mutations that create oncogenes tend to be dominant,mutations in tumor suppressor genes are usually recessive. This has led to the two-hit model

of carcinogenesis (Fig. 6.7).47 Briefly, recessive mutations occur in tumor suppressor genes

but are latent because of the persistence of a wild type allele. The tumor suppressor

phenotype, therefore, requires mutation or loss of the second allele, a process known as loss

of heterozygosity . Alternatively, the second allele of a tumor suppressor gene can be

silenced epigenetically without a direct genetic alteration. A number of genes encoding

negative regulators of the cell cycle conform to this two-hit paradigm.



















Table 6.1. Cell-Cycle Genes Commonly Mutated or Altered in Expression in Human

Cancer

Figure 6.7. The two-hit model of tumor suppression.

Tumor suppressors are proteins that are thought to provide protection from malignancy.

Depicted is a chromosome carrying a tumor suppressor–encoding locus shown in white. At

birth, normal individuals carry two wild type alleles (yellow bands) at tumor suppressor loci.

Over time, however, spontaneous mutations occur at these loci (red flash) that render one

allele nonfunctional (orange band ). However, because such mutations are expected to be

recessive to the wild type allele that is still present, there is no phenotypic consequence.Over time, additional events can lead to loss of the wild type allele, a phenomenon referred

to as loss of heterozygosity (LOH). LOH then provides a tangible contribution to the

malignant phenotype. However, because spontaneous mutations at specific loci and specific

secondary allelic losses are rare events, malignancy usually develops only after a very long

latency period. On the other hand, some individuals are born with inherited tumor

suppressor mutations. Because only LOH is then required for expression of the tumor

suppressor–null phenotype, cancer with decreased latency and higher penetrance develops

in such individuals.

In theory, to achieve uncontrolled cell division, two basic requirements must be met. First,

cells need a strong constitutive proliferation signal capable of overriding the environmental

and internal restraints on division that normal cells experience. Second, the barrier of














senescence needs to be dismantled to render tumor cells immortal. Mutations in a large

variety of cell-cycle control and related genes are associated with malignancy, and most of

these can be accommodated within this framework. This model of tumorigenesis has been

confirmed in rodent tissue culture–based in vitro models. Transfection of primary rodent

fibroblasts with individual plasmids programmed to express proteins that promote eithergrowth or immortalization does not result in malignant transformation. However,

cotransfection of two plasmids, one in each category, does promote transformation

(Fig. 6.8). However, these results need to be interpreted cautiously in the context of human

cancer because immortalization of rodent cells in culture most likely does not involve

telomeres, which are much longer in rodents than in humans.48 One idea that has emerged

is that strong growth signals and other environmental pressures exerted on premalignant

cells produce potent stress responses, leading to cell-cycle blockade or cell

death.49Phenotypically, such stress-induced effects on fibroblasts closely resemble those

associated with replicative senescence; therefore, this phenomenon has been termed stress-

induced senescence (see following discussion). Therefore, genetic alterations are likely

required to neutralize these stress responses to immortalize rodent cells. Transformation of

human cells requires these same genetic alterations, but also telomere attrition must be

reckoned with, requiring additional mutations.

Figure 6.8. Malignant transformation requires multiple genetic alterations.

Depicted is an in vitro experiment using primary rodent embryonic fibroblasts. Cells

transfected with a plasmid programmed to express an activated Ras allele eventually grow to

form a confluent monolayer, at which time proliferation ceases because of inhibition

mediated by cell-cell contact. Similarly, cells transfected with a plasmid programmed to

express a dominant-negative allele of p53 (encoding a protein that can complex with and

inactivate endogenous wild type p53) from a confluent monolayer. However, cells transfected

simultaneously with both plasmids from a confluent monolayer, out of which grow

transformed foci. These piles of cells are no longer subject to the controls that restrict

fibroblast proliferation and, as such, resemble cancer cells. The requirement for two

perturbations in this system supports a mechanism whereby activated Ras stimulates growth

and proliferation, and dominant negative p53 inactivates stress pathways that would cause

these cells to have a limited proliferative lifespan.

Alterations in Pathways Affecting Growth and Proliferation

Mutations that regulate cell growth and proliferation can occur at many levels, ranging from

cell surface receptor–mediated signaling pathways that control proliferation to elements of

the core cell-cycle machinery itself.

Growth and Proliferation Signaling Pathways


















Because a large number of receptors and pathways can influence cell proliferation, many

mutations in elements of these pathways have been recovered in human malignancies. Only

a few examples are cited here. One way to provide a strong constitutive proliferation signal

is to overexpress or deregulate growth factor receptors. HER2/neu, a transmembrane

tyrosine kinase receptor found on many epithelial cell types, is often overexpressed becauseof gene amplification in breast and other cancers.50Presumably the amplitude of proliferation

signaling is abnormally high or completely deregulated in such tumors. Similarly, signaling

elements downstream of mitogen receptors can be mutated to produce constitutive

signaling. Perhaps the best-known example is the case of the Ras family guanosine

triphosphatases, which serve as signal transducers for a number of key proliferation

pathways. Dominant mutations in Ras isoforms that stabilize the activated state confer

strong constitutive proliferation signaling. One of the pathways stimulated by Ras is the PI3-

kinase pathway. A PI3 phosphatase, PTEN, normally reverses this phosphorylation, keeping

the signal in check. Consistent with this, mutational loss of PTEN similarly to oncogenic

mutations in Ras can lead to constitutive signaling contributing to carcinogenesis.

Cell-Cycle Machinery

Signaling pathways that stimulate proliferation impinge on the cell-cycle machinery by

stimulating the biosynthesis of D-type cyclins and promoting the degradation of CDK

inhibitors. Accumulation of D-type cyclins and concomitant activation of CDK4 and CDK6

have been shown to activate the cell-cycle program primarily by phosphorylation and

inactivation of the retinoblastoma protein, pRb, and related proteins p107 and p130. These

proteins form potent repression complexes with transcription factors that are critical for S-

phase entry and progression, notably the E2F family, effectively blocking cell-cycle

progression. In addition, INK4 family inhibitors specifically down-regulate CDK4 and CDK6,

buffering their capacity to phosphorylate pRb and related proteins. Virtually all components

of this pathway have been found to be misregulated or mutated in cancer to provide a

constitutive proliferation signal (proteins with asterisks in Fig. 6.6).51 The genes encoding D-

type cyclins are found amplified in a broad spectrum of tumors. On the other hand, the gene

encoding the INK4 inhibitor, p16, is mutated and lost in some types of cancer, whereas

CDK4 has been found to be mutated so as not to bind p16. In many instances p16, although

not genetically altered, is down-regulated at the epigenetic level. The p16 promoter contains

a CpG island that is subject to repression via methylation. Finally, pRb is the tumor

suppressor on which the so-called two-hit hypothesis was originally formulated. Inherited

mutations in the RB gene and subsequent loss of heterozygosity invariably lead to childhood

retinoblastoma and eventually other malignancies. However, somatic mutation of RB1 and

loss of heterozygosity are found in many sporadic noninherited cancers, underscoring the

critical nature of negative cell-cycle regulation by pRb.

Like D-type cyclins, cyclin E is frequently found up- regulated in cancer. The fact that

deregulated expression of cyclin E can drive cells prematurely into S phase suggests that

cyclin E provides a growth/division stimulus during carcinogenesis. Furthermore, cells from

cyclin E nullizygous mice are resistant to malignant transformation in tissue culture

models.12 However, other evidence suggests that deregulation of cyclin E may promote

carcinogenesis principally by inducing genomic instability rather than by promoting growth



















(see ―Mutations Causing Genetic and Genomic Instability‖ ). Likewise, the CDK2 inhibitor

p27Kip1 is often found down-regulated in cancer, although never behaving as a classic tumor

suppressor inactivated through mutation and allelic loss. However, as with cyclin E

deregulation, it is not clear whether low p27 levels have an impact on carcinogenesis by

promoting growth or genomic instability. While the CDK-inhibitory functions of p27 arerestricted to the nucleus, hyperphosphorylation leads to cytoplasmic translocation, where

non−CDK-bound p27 promotes cell migration. This may explain why p27 is rarely deleted in

cancer, as cytoplasmic functions may be important for invasion and metastasis.52

Alterations in Pathways Affecting Senescence

In addition to a constitutive growth stimulus that overrides natural restraints, tumors need to

have the capacity for unlimited proliferation. Normally, the limited lifespan of somatic cells

imposed by the process of replicative senescence constitutes a natural barrier to

tumorigenesis. Therefore, genes that mediate senescence are commonly mutated in cancer.

However, the issue of senescence is complicated by functional overlap between senescencepathway genes and oncogenic stress pathway genes that also require

inactivation.48,49 Because senescence is a result of checkpoint responses to acute telomere

attrition, genes that encode DNA checkpoint signaling elements and transducers are

targeted. One of the most commonly mutated genes in human cancer encodes the

checkpoint effector p53.36 Inherited mutations in TP53, the gene encoding p53, confer a

syndrome known as Li-Fraumeni characterized by early-onset cancer.53 However, the

majority of sporadic cancers are also mutated at the p53 locus. The role of p53 mutation in

cancer as a promoter of immortalization is supported by the finding that cells from p53

nullizygous mice are immortal.54 However, this conclusion is complicated by the fact that

p53 is central to cellular stress responses that also require inactivation during malignant

transformation, and as previously stated, telomere attrition is not likely to be a significant

issue for immortalization in mice. Nevertheless, an observation supporting the idea that

checkpoint genes likely to be triggered by telomere attrition are targeted to immortalize

premalignant cells is that chk2, a signaling element of the DNA damage checkpoint response,

is mutated in a subset of patients with Li-Fraumeni syndrome53rather than p53.

Mutation of the gene encoding Nbs1, required for activation of chk1 and chk2 kinases, is also

associated with a hereditary cancer syndrome,55 Nijmegen disease, as well as sporadic

cancers, although the interpretation of this result is complicated by the fact that Nbs1 is also

involved in DNA damage repair (see ―Mutations Causing Genetic and Genomic Instability‖ ).

However, the most direct strategy to bypass senescence is to induce directly the expression

of telomerase in somatic cells. c-Myc, a transcription factor linked to stimulation of

proliferation, has also been shown to be a positive regulator of the gene encoding telomerase

reverse transcriptase (hTERT), the catalytic subunit of telomerase.56 This may explain the

high frequency of human tumors exhibiting c-Myc amplification or overexpression, or both.

However, there appear to be a number of different mutational targets that can lead to

derepression of the hTERT gene.57

Mutations Neutralizing Stress Responses

















































model and, consistent with this idea, cyclin E deregulation led to accelerated loss of

heterozygosity at the TP53 locus (encoding p53), which correlated with higher tumor

incidence.65 Interestingly, an essential component of the ubiquitin ligase responsible for cell

cycle–dependent targeting of cyclin E for proteolysis, hCDC4 (also known as FBXW7), is often

found mutated in cancer,27,29,66 and its deletion has been shown to also cause genomicinstability in cultured cells.67 Thus, genetic alterations that interfere with proper regulation

of cell-cycle machinery have the potential of affecting not only the cell cycle itself, but also

the genetic and genomic integrity of the cell.

MicroRNAs, the Cell Cycle, and Cancer

Although discovered in the nematode Caenorhabditis elegans many years ago, the

importance of microRNAs (miRs) in mammalian cells and human cancer has only recently

begun to be appreciated.68,69These small RNAs target specific mRNAs via degradation or

inhibition of translation. They confer unique regulatory possibilities in that they usually target

several different mRNAs simultaneously, allowing for coordination of multiple pathways. At

least five groups or clusters of miRs have been shown to target mRNAs encoding cell-cycle

regulatory proteins. The miR-15a/16 cluster targets cyclin E1, cyclin D1, and cyclin D3, as

well as CDK6. Not surprisingly, this miR cluster has been shown to have tumor suppressive

functions in several different types of cancer. The miR-17/20 cluster, which targets cyclin D1

and E2F transcription factors, and let-7, which targets cyclin D1, cyclin D3 cyclin A, CDK4,

and CDK6, have been also been shown to be tumor suppressive. Conversely, the miR-221-

222 cluster and mir-21, which target a number of CDK inhibitors, has oncogenic properties.

Understanding of the regulation and roles of miRs in normal cell function and cancer etiology

is largely incomplete, and filling in the gaps poses a significant research challenge for thecoming years.

The Cell Cycle and Cancer Therapy

Because cancer cells must proliferate, essential cell-cycle proteins have been suggested as

targets for therapeutic exploitation. Notably, CDKs have been extensively screened for small-

molecule inhibitors, some of which are in clinical trials. It is too early, however, to judge the

efficacy of this approach beyond its success using in vitro models. An alternative approach

being explored is to develop agents that undermine checkpoint responses. The presumption

is that cancer cells, because of their highly proliferative state, might be more susceptible to

loss of essential controls. This idea remains to be confirmed. However, it is noteworthy that

many therapeutic approaches currently use compounds that normally trigger checkpoint

responses, such as genotoxic agents or spindle poisons. It is assumed that these treatments

are effective because tumor cells are actually impaired in their defensive checkpoint

responses. An interesting approach that initially showed promise in model systems but has

proven disappointing in clinical trials, uses the fact that a large percentage of malignancies

are defective for p53 function in order to evade checkpoint and stress responses. A common

human lytic virus known asadenovirus, expresses an essential gene, E1B p55K, specifically to

down-regulate p53 in order to allow a productive infection. Oncolytic adenoviruses have

therefore been engineered to not express E1B p55K.70 These adenoviruses are harmless tonormal cells but can productively infect and lyse p53-defective tumor cells in tissue culture
































and mouse xenograft models. However, technical issues such as low tumor infectivity, rapid

viral clearance, and neutralizing immune responses in clinical trials have limited the efficacy

of this approach.70 On the other hand, if new generations of oncolytic viruses that

circumvent these problems can be developed, this may constitute one of the more promising

new therapeutic approaches.

Circadian rhythms may present another interesting link between cancer therapeutics and the

cell cycle. Although circadian rhythms regulate virtually every aspect of human physiology, it

is clear that cell-cycle regulatory gene expression and the cell cycle itself are entrained to the

day-night cycle.71 It has also been shown that tumor cells have largely lost this regulation.

This difference, therefore, may be exploitable therapeutically. Indeed, the tolerance and

efficacy of a variety of genotoxic chemotherapeutic agents varies with time of day,

suggesting a possible link between the time of administration and the cell-cycle position of

cells in healthy tissues. More detailed understanding of circadian regulation of the cell cycle

may provide an avenue for optimization of current therapeutics.

References

1. Howard A, Pelc SR. Nuclear incorporation of p32 as demonstrated by

autoradiographs. Exp Cell Res 1951;2:178.

2. Johnson RT, Rao PN. Mammalian cell fusion: induction of premature chromosome

condensation in interphase nuclei. Nature 1970;226:717. [PMID: 5443247]

3. Rao PN, Johnson RT. Mammalian cell fusion: studies on the regulation of DNA

synthesis and mitosis. Nature 1970;225:159. [PMID: 5409962]

4.

Masui Y, Markert CL. Cytoplasmic control of nuclear behavior during meioticmaturation of frog oocytes. J Exp Zool 1971;177:129. [PMID: 5106340]

5. Hartwell LH, Culotti J, Pringle JR, et al. Genetic control of the cell division cycle in

yeast. Science1974;183:46. [PMID: 4587263]

6. Harper JW, Adams PD. Cyclin-dependent kinases. Chem Rev 2001;

101:2511. [PMID: 11749386]

7. Jeffrey PD, Russo AA, Polyak K, et al. Mechanism of CDK activation revealed by the

structure of a cyclinA-CDK2 complex. Nature 1995; 376:313. [PMID: 7630397]

8. Stevens C, La Thangue NB. E2F and cell cycle control: a double-edged sword. Arch

Biochem Biophys 2003;412:157. [PMID: 12667479]

9. Stiegler P, Giordano A. The family of retinoblastoma proteins. Crit Rev Eukaryot

Gene Expr 2001;11:59. [PMID: 11693966]

10. Ohtsubo M, Roberts JM. Cyclin-dependent regulation of G1 in mammalian

fibroblasts. Science1993;259:1908. [PMID: 8384376]

11. Resnitzky D, Gossen M, Bujard H, et al. Acceleration of the G1/S phase transition by

expression of cyclins D1 and E with an inducible system. Mol Cell

Biol 1994;14:1669. [PMID: 8114703]

12. Geng Y, Yu Q, Sicinska E, et al. Cyclin E ablation in the mouse. Cell 2003;

114:431. [PMID: 12941272]

13. Berthet C, Aleem E, Coppola V, et al. CDK2 knockout mice are viable. CurrBiol 2003;13:1775.[PMID: 14561402]




http://www.ncbi.nlm.nih.gov/pubmed/5443247?dopt=Abstract



















































14. Ortega S, Prieto I, Odajima J, et al. Cyclin-dependent kinase 2 is essential for

meiosis but not for mitotic cell division in mice. Nat Genet 2003; 35:25. [PMID:

12923533]

15. Aleem E, Kiyokawa H, Kaldis P. Cdc2-cyclin E complexes regulate the G1/S phase

transition. Nat Cell Biol 2005;7:831. [PMID: 16007079] 16. Polanowska J, Fabbrizio E, Le Cam L, et al. The periodic down regulation of cyclin E

gene expression from exit of mitosis to end of G(1) is controlled by a deacetylase-

and E2F-associated bipartite repressor element. Oncogene 2001;20:4115. [PMID:

11464278]

17. Fung TK, Poon RY. A roller coaster ride with the mitotic cyclins. Semin Cell Dev

Biol 2005;16:335.[PMID: 15840442]

18. Russo AA, Jeffrey PD, Pavletich NP. Structural basis of cyclin-dependent kinase

activation by phosphorylation. Nat Struct Biol 1996;3:696. [PMID: 8756328]

19. Russo AA, Tong L, Lee JO, et al. Structural basis for inhibition of the cyclin-

dependent kinase CDK6 by the tumour suppressor

p16INK4a. Nature 1998;395:237. [PMID: 9751050]

20. Cheng M, Olivier P, Diehl JA, et al. The p21(Cip1) and p27(Kip1) CDK inhibitors are

essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO

J 1999;18:1571. [PMID: 10075928]

21. Russo AA, Jeffrey PD, Patten AK, et al. Crystal structure of the p27Kip1 cyclin-

dependent-kinase inhibitor bound to the cyclin A-Cdk2

complex. Nature 1996;382:325. [PMID: 8684460]

22. Yang J, Bardes ES, Moore JD, et al. Control of cyclin B1 localization through

regulated binding of the nuclear export factor CRM1. GenesDev 1998;12:2131. [PMID: 9679058]

23. Nasmyth K. Disseminating the genome: joining, resolving, and separating sister

chromatids during mitosis and meiosis. Annu Rev Genet 2001;35:673. [PMID:

11700297]

24. Reed SI. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nat

Rev Mol Cell Biol 2003;4:855. [PMID: 14625536]

25. Carrano AC, Eytan E, Hershko A, et al. SKP2 is required for ubiquitin-mediated

degradation of the CDK inhibitor p27. Nat Cell Biol 1999; 1:193. [PMID: 10559916]

26. Tedesco D, Lukas J, Reed SI. The pRb-related protein p130 is regulated by

phosphorylation-dependent proteolysis via the protein-ubiquitin ligase

SCF(Skp2). Genes Dev 2002;16:2946.[PMID: 12435635]

27. Strohmaier H, Spruck CH, Kaiser P, et al. Human F-box protein hCdc4 targets cyclin

E for proteolysis and is mutated in a breast cancer cell

line. Nature 2001;413:316. [PMID: 11565034]

28. Koepp DM, Schaefer LK, Ye X, et al. Phosphorylation-dependent ubiquitination of

cyclin E by the SCFFbw7 ubiquitin ligase. Science 2001;294:173. [PMID: 11533444]

29. Moberg KH, Bell DW, Wahrer DC, et al. Archipelago regulates cyclin E levels in

Drosophila and is mutated in human cancer cell lines. Nature 2001;413:311. [PMID:

11565033]











































































30. Davis RJ. Transcriptional regulation by MAP kinases. Mol Reprod

Dev 1995;42:459. [PMID: 8607977]

31. Chang F, Lee JT, Navolanic PM, et al. Involvement of PI3K/Akt pathway in cell cycle

progression, apoptosis, and neoplastic transformation: a target for cancer

chemotherapy. Leukemia2003;17:590. [PMID: 12646949] 32. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the

nucleus. Cell 2003;113:685. [PMID: 12809600]

33. Reynisdottir I, Polyak K, Iavarone A, et al. Kip/Cip and Ink4 Cdk inhibitors cooperate

to induce cell cycle arrest in response to TGF-beta. Genes Dev 1995;9:1831. [PMID:

7649471]

34. Elledge SJ. Cell cycle checkpoints: preventing an identity

crisis. Science 1996;274:1664. [PMID: 8939848]

35. Yang J, Yu Y, Hamrick HE. ATM, ATR, and DNA-PK: initiators of the cellular genotoxic

stress responses. Carcinogenesis 2003;24:1571. [PMID: 12919958]

36. Vousden KH. Activation of the p53 tumor suppressor protein. Biochim Biophys

Acta 2002;1602:47.[PMID: 11960694]

37. Taylor WR, Stark GR. Regulation of the G2/M transition by

p53. Oncogene 2001;20:1803. [PMID: 11313928]

38. Bartek J, Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer

Cell 2003;3:421. [PMID: 12781359]

39. Sorensen CS, Syljuasen RG, Falck J, et al. Chk1 regulates the S phase checkpoint by

coupling the physiological turnover and ionizing radiation-induced accelerated

proteolysis of Cdc25A. Cancer Cell 2003;3:247. [PMID: 12676583]

40. Allshire RC. Centromeres, checkpoints and chromatid cohesion. Curr Opin GenetDev 1997;7:264.[PMID: 9115433]

41. Blagosklonny MV, Pardee AB. The restriction point of the cell cycle. Cell

Cycle 2002;1:103. [PMID: 12429916]

42. Ekholm SV, Zickert P, Reed SI, et al. Accumulation of cyclin E is not a prerequisite

for passage through the restriction point. Mol Cell Biol 2001;21:3256. [PMID:

11287628]

43. Martinsson HS, Starborg M, Erlandsson F, et al. Single cell analysis of G1 check

points—the relationship between the restriction point and phosphorylation of

pRb. Exp Cell Res 2005;305:383.[PMID: 15817163]

44. Smith JR, Pereira-Smith OM. Replicative senescence: implications for in vivo aging

and tumor suppression. Science 1996;273:63. [PMID: 8658197]

45. Harley CB, Sherwood SW. Telomerase, checkpoints and cancer. Cancer

Surv 1997;29:263. [PMID: 9338104]

46. Woo RA, Poon RY. Cyclin-dependent kinases and S phase control in mammalian

cells. Cell Cycle2003;2:316. [PMID: 12851482]

47. Knudson AG Jr. Hereditary cancer. JAMA 1979;241:279. [PMID: 758536]

48. Sherr CJ, DePinho RA. Cellular senescence: mitotic clock or culture

shock? Cell 2000;102:407.[PMID: 10966103]

49. Schmitt CA. Cellular senescence and cancer treatment. Biochim Biophys

Acta 2007;1775:5. [PMID: 17027159]





















































































chapter 6 - cell cycle

Documents