Cell Cycle

Elisa Ventura, Temple University, Philadelphia, PA, United States

Antonio Giordano, Temple University, Philadelphia, PA, United States and University of Siena, Siena, Italy
r 2019 Elsevier Inc. All rights reserved.

Glossary
Cell cycle checkpoints Cell cycle checkpoints are control
mechanisms involving different groups of molecules that act
at critical stages of the cell cycle, i.e., the G1, G1/S, S and G2
or G2/M stages, and, by integrating signals coming from
both inside and outside the cells, allow the transition to the
next phase of the cell cycle only in case the conditions are
favorable and all processes have been properly completed.
Otherwise, depending on the severity of the sensed defects/
alterations, they halt the cell cycle in a transient or
permanent manner or trigger apoptosis.
Cell cycle The cell cycle is the sequence of cell duplication
and division. The cell cycle can be divided into four phases
known as M, during which the division of the nucleus
(mitosis) and the cytoplasm (cytokinesis) occurs, S,
characterized by DNA duplication, G1 and G2, during
which cell growth takes place.
Cyclin-dependent kinase inhibitors (CDKi) CDK
inhibitors are molecules belonging to the INK4 or Cip/kip
protein families. By inhibiting CDKs, they function as cell
cycle restrainers.
Cyclin-dependent kinases (CDKs) CDKs form a family of
proline-directed Ser/Thr kinases including twenty one
members. Some CDKs, known as cell cycle CDKs, are the
master regulators of the cell cycle. Indeed, by interacting
with their regulatory subunits, cyclins, phosphorylate, in a
cyclic manner, specific sets of molecules allowing the correct
and ordered sequence of events leading to cell cycle
progression.
Cyclins Cyclins are a group of proteins whose levels
oscillate along the cell cycle thanks to their periodic
expression and controlled degradation. By interacting with
CDKs, cyclins activate and address CDKs to specific sets of
substrates driving cell cycle progression.
Cytokinesis Cytokinesis is the process of cytoplasm
division.
G1 restriction point The G1 restriction point is a G1 stage
marked by the inactivation of the retinoblastoma proteins at
which cells are committed to divide independently on the
presence of mitogenic stimuli.
Interphase Interphase is the interval lasting between two
consecutive mitosis. It includes the G1, S and G2 phases.
Mitosis From the Greek word “mitos” which means
“thread”, mitosis is the division of the nucleus.

Historical Perspective of Cell Cycle Regulation

The landmark studies leading to the understanding of cell cycle regulation date back to the 1980s and to the pioneering work of
Nobel Laureates Lee Hartwell, Tim Hunt and Paul Nurse who, by studying cell division in budding yeast, sea urchin and fission
yeast, uncovered the basis of cell cycle regulation and showed that the key genes and mechanisms responsible for cell cycle control
are highly conserved in eukaryotes, from yeast to humans (Yanagida, 2014).
The history of the study of the cell cycle started in 1880 with Flemming and Strasburger who, by observing at the optic
microscope sections of onion root pits, introduced the concept of mitosis (from the Greek word “mitos” which means “thread”, to
describe the nuclear structures that are visible at the beginning of mitosis and that we now know to be condensed chromosomes)
to indicate nuclear division (Paweletz, 2001). Until the first half of the twentieth century, the cell cycle was thus described as a two
phases process made of the M-phase, characterized by nuclear and cytoplasmic (cytokinesis) division and the interphase, defined
as the interval lasting between two consecutive mitosis. In 1951, Howard and Pelc, by incubating bean root tips with radioactive
phosphorus, observed that DNA replication occurs in discrete periods of time. They thus introduced the concept of the DNA
synthesis (S) phase, during which the duplication of the genetic material occurs (Howard and Pelc, 1951). These observations led
to the still valid descriptive model of the cell cycle as divided into four phases, the S and M phases, and the two gap (G) phases
known as G1 and G2, preceding the S and M phases, respectively, during which cell growth occurs (Fig. 1).
Twenty years after Howard and Pelc’s observations, further studies described how the cell cycle is regulated and the key
molecules that allow the correct and ordered progression through the G1, S, G2 and M phases, preventing the cell from moving
back in the cell cycle. Indeed, in 1970, Rao and Johnson, by fusing cells arrested in different cell cycle phases could demonstrate
the existence of dominant factors able to initiate and drive a specific cell cycle phase. As an example, they observed that by fusing
cells arrested in G1, S or G2 with cells in mitosis, processes typical of early mitosis, such as nuclear membrane vesiculation and
chromosome condensation, were induced in G1-, S- or G2-arrested cells. They thus hypothesized the existence of a diffusible factor
that they called “the M-phase inducer”, that, once transferred from mitotic cells to G1-, S- or G2- arrested cells, was able to trigger
mitosis (Johnson and Rao, 1970). Based on analogous observations, they also hypothesized the existence of factors inducing DNA
synthesis and delaying or blocking the cells in G1 or G2 phases (Rao and Johnson, 1970). In the same years, Masui & Market
demonstrated that it was possible to initiate meiosis in frog oocytes (G2-arrested cells), upon the injection into oocytes of

Reference Module in Life Sciences doi:10.1016/B978-0-12-809633-8.90189-4 1

2 Cell Cycle

Fig. 1 Classical model of the cell cycle. The cell cycle consists of four phases: the S-phase, characterized by DNA duplication, the M-phase,
characterized by nucleus (mitosis) and cytoplasm (cytokinesis) division, and the two gap (G) phases, G1 and G2, during which cell growth occurs.
Non proliferating cells as well as cells that are not subjected to mitogenic stimuli enter a quiescent state known as G0, from which they can
reenter the cell cycle upon proper mitogenic stimuli. Each phase of the cell cycle is driven by a specific cyclin-dependent kinase (CDK)-cyclin
complex that, by phosphorylating specific sets of molecules in a timely fashion, ensures the correct progression trough the cell cycle preventing
the cell from moving back in the cell cycle. The phosphorylation and the consequent inactivation of the retinoblastoma protein family members
RB1/p105, RBL1/p107 and RBL2/p130 mark the so called G1 restriction point, a stage after which cells are committed to divide independently on
the presence of mitogenic stimuli. Regulatory molecules including the kinase Wee1, the phosphatases cdc25, the CDK inhibitors p15, p16, p18,
p19, p21, p27 and p57 and checkpoints mechanisms integrate intracellular and extracellular signals and, by affecting the activity of CDK-cyclin
complexes, allow the progression of the cell cycle and the transition to the next phase only in case the conditions are suitable.

cytoplasm collected from frog eggs (meiosis-arrested cells), and that this could be done by serial transfers. They hypothesized that
this was possible because of the presence of a “Maturation-promoting factor” in frog eggs (Masui and Markert, 1971). Similar
experiments were performed in starfish oocytes and eggs, in mouse oocytes, in mammalian cells and in yeast, bringing to the
concept of the existence of a universal factor, called “M-phase-promoting factor (MPF)”, able to initiate mitosis/meiosis (Prigent
and Hunt, 2004 and references therein). The next important step in the identification of the MPF was its purification. Maller’s
group fractionated the cytoplasm derived from frog eggs and identified the fraction that was able to induce, in vitro, the nuclear
division of frog sperm-derived nuclei. They could then purify from the identified fraction a heterodimer, made of two proteins of
32 and 45 kDa, whose activity was high in mitosis and low in interphase, and which showed M-phase-promoting and kinase
activity in vitro (Lohka et al., 1988).
In 1983, by using sea urchin as a model, proteins whose levels changed in a cyclic manner depending on the cell cycle phase,
were identified and named “cyclins” by Hunt’s group (Evans et al., 1983; Evans, 2004). The injection of cyclin mRNA into frog
oocytes was able to induce their maturation into frog eggs. Also, cyclin mRNA, when added in vitro to frog sperm-derived nuclei, in
the presence of RNA-depleted frog eggs cytoplasm, was sufficient to induce nuclear mitotic entry. At the same time, Hartwell and
Nurse, by using cell division cycle (cdc) mutant yeasts, identified a gene encoding for a kinase showing high activity in mitosis and
low activity in interphase, which was named cdc2 in the fission yeast Schizosaccharomyces pombe and Cdc28 in the budding yeast
Saccharomyces cerevisiae (Beach et al., 1982; Wood and Hartwell, 1982; Hartwell and Smith, 1985). Additional key experiments
allowed to put the pieces of the puzzle together and to identify in cdc2 and cyclin B the two MPF components of 32 and 45 kDa,
respectively, and thus to identify in the complex cdc2-cyclin B the key driver of mitosis (Dunphy et al., 1988; Gautier et al., 1988;
 Cell Cycle 3

Draetta et al., 1989; Labbé et al., 1989; Meijer et al., 1989; Gautier et al., 1990). Further studies led to the identification of other
kinases active in different phases of the cell cycles and to the description of the basic model of cell cycle regulation, according to
which each phase of the cell cycle is triggered by a specific CDK-cyclin complex (Fig. 1), as described in the next paragraphs.

Cell Cycle Key Molecular Regulators: Cyclin-Dependent Kinases (CDKs), Cyclins and CDK Inhibitors

CDKs and Cyclins

CDKs are proline-directed serine/threonine protein kinases playing a central role in the control of cell cycle progression
(Malumbres, 2011, 2014). CDKs phosphorylate a broad range of proteins that initiate and regulate the various events that
characterize each phase of the cell cycle (Graña and Reddy, 1995). Since the activity of CDKs oscillates during the cell cycle, the
activity of CDK substrates varies in a cyclical manner allowing the correct and ordered sequence of events leading to cell cycle
progression (Graña and Reddy, 1995). The cyclic activity of CDKs is regulated by a complex network of proteins and enzymes
and primarily by CDKs transient association with cyclins, that activate and direct CDKs on specific substrates in a timely manner
(Obaya and Sedivy, 2002). Indeed, the levels of the various CDKs are quite constant along the cell cycle, whereas the levels of
the different cyclins oscillate thanks to their periodic synthesis and controlled degradation. In yeast, the progression trough the
cell cycle is dependent on only one CDK, the above mentioned Cdc2 in Schizosaccharomyces pombe and Cdc28 in Saccharomyces
cerevisiae, that interacts with different cyclins depending on the cell cycle phase (Gérard et al., 2015). Human genome contains
many genes encoding for CDKs, CDK-like proteins and for proteins containing a ‘‘cyclin box’’ and thus classified as cyclins (21, 5
and 29, respectively), but only some of them are involved in the regulation of the cell cycle (Malumbres and Barbacid, 2005;
Malumbres et al., 2009). Based on their functions, CDKs may be divided into two main sub-groups: cell cycle CDKs (CDK1,
CDK2, CDK4, CDK6) and transcriptional CDKs (principally CDK7, CDK8, CDK9). Concerning cyclins, the members of the A
(A1 and A2), B, D (D1, D2 and D3) and E (E1 and E2) classes of cyclins are directly involved in the control of cell cycle
progression.

Cell Cycle Regulation by CDK-Cyclin Complexes

Human adult tissues are mainly composed by cells that have entered a quiescent state, known as G0. For some cell types, such as
neurons and skeletal muscle cells, this condition, known as terminally differentiated G0 state, is usually irreversible since these
cells are not able to divide anymore and all the machinery necessary for cell cycle progression is switched off. Other cell types enter
the G0 state only transiently and can physiologically re-enter to early G1 upon mitogenic stimuli (Ren and Rollins, 2004).
Mitogens trigger different signaling pathways and principally the Ras-dependent mitogen-activated protein kinase (MAPK) cascade
and the phosphatidyl inositol 3 kinase (PI3K)-AKT pathway, that converge on the activation of the CDKs that govern G1
progression, i.e., the two highly homologous CDK4 and CDK6. Indeed, mitogenic stimuli promote the expression and stabili-
zation of D-type cyclins (D1, D2 and D3), as well as their association with CDK4 and CDK6, leading to CDK4/CDK6 activation
(Peeper and Bernards, 1997; Marshall, 1999; Ewen, 2000; Cheng et al., 1998; Diehl and Sherr, 1997; Diehl et al., 1998, 2003;
Baldin et al., 1993). Also, signaling pathways mediated by the T-cell receptor, cytokine and hormone receptors as well as by cell
adhesion molecules regulate the levels of D-type cyclins and thus affect the cell cycle in specific cell types (Sherr et al., 2016).
Activated CDK4 and CDK6 phosphorylate and partially inactivate the three members of the retinoblastoma (RB) protein family, i.e
RB1/p105, retinoblastoma-like (RBL)1/p107 and retinoblastoma-like (RBL)2/p130 (Buchkovich et al., 1989; Chen et al., 1989;
DeCaprio et al., 1989; Mihara et al., 1989; Harbour et al., 1999; Ewen et al., 1993; Kato et al., 1993; Lundberg and Weinberg, 1998;
Genovese et al., 2006; Fig. 1). RB proteins form, in quiescent state, complexes with the E2F transcription factors. RB proteins partial
inactivation determines the release of the E2F transcription factors that in turn promote the expression of G1/S genes and in
particular of G1/S-cyclins (cyclins E) and S-cyclins (cyclins A) (Dyson et al., 1989; Trimarchi and Lees, 2002; Giacinti and
Giordano, 2006; Indovina et al., 2013; Sun et al., 2007). Also, the E2F transcription factors promote their own expression in a
positive feedback loop that favors the irreversible entry into G1 (Bertoli et al., 2013). The inactivation of RB proteins by CDK4/
CDK6 denotes a stage known as restriction point: cells overcoming the restriction point are committed to divide independently on
the presence of extracellular mitogenic stimuli (Bartek et al., 1996). In addition to the RB proteins, CDK4 and CDK6 also activate
the forkhead box protein M1 (FOXM1) which regulates the expression of genes involved in the control of cell cycle progression
(Anders et al., 2011). E-type cyclins bind to and activate CDK2 which, in turn, phosphorylates RB proteins leading to their full
inactivation (Lundberg and Weinberg, 1998; Harbour et al., 1999; Akiyama et al., 1992). In addition to RB proteins, CDK2
substrates include other proteins that promote cell cycle progression (the CDK inhibitor p27) (Sheaff et al., 1997), entry in S-phase
(the nuclear protein mapped to the ATM locus (NPAT)) (Ma et al., 2000), chromosome de-condensation (histone H1) favoring
fork progression (Alexandrow and Hamlin, 2005), DNA synthesis (DNA polymerase alpha primase and the replication factors A
and C) (Voitenleitner et al., 1997), and centrosome duplication (nucleophosmin, NPM) (Okuda et al., 2000). In the late stage of
the S-phase, CDK2 associates with cyclin A (A2 in somatic cells and A1 in germ cells) in turn addressing CDK2 to targets whose
activation determines the transition to the G2-phase. In the G2-phase, cyclin A2 binds to and activates CDK1, promoting the
phosphorylation of the transcription factors FOXM1 and FOXK2 that control the expression of genes crucial for mitotic entry
(Laoukili et al., 2008; Marais et al., 2010). The breakdown of the nuclear membranes regulated by cyclin A2 determines the
4 Cell Cycle

degradation of A-type cyclins and allows CDK1 binding to cyclin B, the last one being localized to the cytoplasm because of the
presence of a nuclear exclusion signal (Gong et al., 2007; Porter and Donoghue, 2003). CDK1-cyclin B are the master regulators of
mitosis: CDK1 phosphorylates more than seventy targets to trigger chromosome condensation, centrosome separation, mitotic
spindle assembly, Golgi dynamics and other central processes of the two first phases of mitosis, i.e., prophase and metaphase
(Ubersax et al., 2003). The progression through anaphase requires a reduction in the activity of CDK1. This is achieved by the
degradation of cyclin B, a process that is mediated by the anaphase-promoting complex/cyclosome (APC/C) (Morgan, 1999).
Mitotic exit is marked by the complete inactivation of CDK1 (Nigg, 2001).
All CDKs also phosphorylate their own regulators Wee1 (Heald et al., 1993) and cdc25 (Hoffmann et al., 1993) and different
components of the cytoskeleton, such as nuclear lamins, vimentin and microtubules, allowing the cytoskeleton reorganizations
crucial for nuclear and cytoplasmic division (Courvalin et al., 1992; Blangy et al., 1995).
Despite the described model of cell cycle regulation is the classical and accepted one, studies performed in mice lacking
single CDKs challenge it, as mice embryos harboring CDK2, CDK4 or CDK6 mutations could survive until mid gestation (Rane
et al., 1999; Tsutsui et al., 1999; Malumbres et al., 2004; Ortega et al., 2003; Berthet et al., 2003). Similar results were obtained in
mice lacking the cyclins D (Kozar et al., 2004; Geng et al., 1999) and E (Geng et al., 2003). By contrast, mice embryos lacking the
mitotic CDK, CDK1, could not develop beyond the two-cell stage (Santamaría et al., 2007). These experiments suggest that only
CDK1 is essential for cell division in the embryo and sufficient to promote the cell cycle, and that functional redundancy may
exist among different CDKs and cyclins. However, the same studies indicate that certain CDKs and cyclins may play an essential
role in specific cell lineage: CDK6 and cyclin A2 in the proliferation of haematologial precursors, CDK4 in pancreatic b-cells cell
division, CDK2 in meiosis and cyclin D3 in lymphocytes development.

CDK Activity Modulation by Kinases, Phosphatases and CDK Inhibitors

In addition to be dependent on cyclin binding, CDK activity is modulated by CDK phosphorylation status. To be active CDKs
must be phosphorylated by a complex known as CDK-activating kinase (CAK) which consists of CDK7, cyclin H and MAT
(ménage a trois) (Larochelle et al., 1998, 2007). By contrast, CDK phosphorylation by the kinases Wee1 and Myt1 leads to
their inactivation. CDK inactivating phosphorylation can be removed by Cdc25 phosphatases (Donzelli and Draetta, 2003 and
references therein). The Cdc25 family includes cdc25A, cdc25B and cdc25C that activate CDKs at the G1/S transition, S-phase
and entry into mitosis, respectively. Also, CDK activity is modulated by the sequestration of different molecules involved in
their regulation in specific cellular compartments. Similarly to B cyclin that localizes to the cytosol to avoid early entry in
mitosis (Yang et al., 2001), Wee1 and Myt1 localize to the nucleus (Heald et al., 1993) and the Golgi and the endoplasmic
reticulum (Liu et al., 1997), respectively, and cdc25 is retained in the cytosol during interphase by the 14-3-3-proteins
(Dalal et al., 1999).
Another key level of regulation of CDK activity is achieved throughout the interaction of CDKs or CDK-cyclin complexes with
molecules collectively known as CDK inhibitors (reviewed by Besson et al. (2008)). CDK inhibitors belong to two different protein
families, known as the INK4 and Cip/Kip families. The INK4 family includes the four proteins p15INK4a, p16INK4b, p18INK4c and
p19INK4d (Sherr and Roberts, 1999). All INK4 proteins act by halting the progression in G1 since they inhibit CDK4 and CDK6, by
preventing their interaction with cyclins D (Fig. 1). INK4 proteins levels are increased in case of lack of mitogens, in response to
anti-proliferating or differentiation stimuli, in response to altered oncogenes or oncosuppressors signaling and during senescence.
Depending on the context, different INK4 proteins are activated. p15 expression is mainly regulated by anti-mitogenic factors such
as transforming growth factor (TGF)-b (Reynisdóttir et al., 1995; Hannon and Beach, 1994). p16 is up-regulated in senescent cells
and its expression is induced by oncogenes and oncosuppressors-related signals as p16 expression is induced by Ras activation and
following RB1/p105 and p53 inactivation (Serrano et al., 1997).
The Cip and Kip family consists of the proteins p21, p27 and p57, encoded by the genes cdkn1a, cdkn1b and cdkn1c, respectively
(Sherr and Roberts, 1999). All Cip/Kip proteins may interfere with the activity of all the CDK-cyclin complexes by interacting with
both the CDK and cyclin components and show a preferential inhibition of CDK2-cyclin E and CDK2-cyclin A in case of p21, and
of CDK2-cyclin E in case of p27. Despite their mechanism of action is similar and they all act by restraining the cell cycle, the
various Cip/kip proteins are activated in response to different signals: p21 and p27 show an ubiquitous expression pattern with
p21 playing a central role in the response to DNA damage insults (Karimian et al., 2016) and p27 in keeping cells in quiescent
states (Besson et al., 2006), whereas p57 has a tissue-restricted expression pattern and has an important role in development
(Andrews et al., 2007; Besson et al., 2004). The binding of p21 and p27 to the various CDK-cyclin complexes and the intensity of
the interaction is modulated by their phosphorylation status (reviewed by Borriello et al. (2007); Child and Mann (2006)). As
examples, the phosphorylation of p21-Thr57 operated by CDK2 or glycogen synthase kinase 3b (GSK3b), enhances its affinity for
CDK1-cyclin B complexes increasing their stability and thus promoting the G2/M transition (Dash and El-Deiry, 2005), whereas
the phosphorylation of other p27 residues by Src, lyn or Abl reduces and increases p27 affinity for CDK2 and CDK4, respectively
(Kardinal et al., 2006; Chu et al., 2007; Grimmler et al., 2007). Also, p21 and p27, depending on the phosphorylation status,
localize to different sub-cellular compartments or are degraded in an ubiquitin-dependent manner (Rodier et al., 2001; Sekimoto
et al., 2004). Finally, p21 and p27 activity may be regulated by their interaction with other molecules. For example, the interaction
of p21 with Set/TAF (Template-activating factor)1 abolishes the inhibition of CDK2-cyclin E and increases the inhibition of CDK1-
cyclin B (Canela et al., 2003).
 Cell Cycle 5

CDKs: Cell Cycle Regulation and other functions

In addition to their key role in regulating the cell cycle, cell cycle CDKs modulate several other processes (reviewed by Lim and
Kaldis (2013)) including gene transcription (CDK2 and CDK1 promote the activity of the transcription factors FOXM1 and
FOXM2), DNA repair (Cyclin E localizes to and stabilizes delayed replication forks and promotes checkpoint kinase 1 (CHK1)
activation, Cyclin D1 accumulates at the site of double strand brakes (DSB) and, along with other factors, promotes DNA repair via
homologous recombination), metabolism (CDK5 mediates glycogen metabolism in liver), stem cell self-renewal (Oct-4 expres-
sion may be regulated by CDK1), epigenetic regulation (the epigenetic factors enhancer of zester homolog 2 (EZH2) and DNA
methyl-transferase 1 (Dnmt1) are phosphorylated and activated by CDK1 and CDK2), T cell activation (CDK4 and CDK6 by
regulating the nuclear factor of activated T cells (NFAT) proteins influence T cells activation), neuronal functions and sperma-
togenesis, even without forming CDK-cyclin complexes and acting as single molecules.
As mentioned above, the CDK family includes several members with associated functions other than cell cycle control and
mainly related to the regulation of basal transcription. However, functions related to cell cycle regulation have also been
described for some of them (reviewed by Malumbres (2014)). CDK3 has a role in RB-dependent G0 cell cycle exit (Ren and
Rollins, 2004). CDK5 plays a role in the regulation of different post-mitotic events in specialized cell types such as neurons.
CDK7, in addition of being involved in basal transcription regulation, is a key component of the CAK complex, as described
above. CDK10 and CDK11, beyond transcription and splicing, modulate G2/M transition. Finally, CDK9 and CDK12 have
important functions in the DNA damage response which is indirectly linked to cell cycle regulation as proper progression
through the cell cycle must satisfy some fundamental criteria such as the presence of intact DNA, as described in the next
section.

Cell Cycle Checkpoints

Proper cell division must satisfy two fundamental requirements: (1) the maintenance of genomic integrity; (2) the main-
tenance of cell nucleo-cytoplasmic ratio within limits that are compatible with cell life. This implies a correct sequence of
events leading to DNA replication, chromosome segregation and cell division, and the coordination of DNA replication with
cell growth, cell mass doubling and organelle duplication (Novák et al., 2018; Tyson and Novak, 2008). Cells evolved a fine-
tuned molecular control system to ensure the correct sequential activation and inactivation of the sets of molecules that govern
each phase of the cell cycle, integrating signals coming from both outside and inside the cell. Control mechanisms known as
“cell cycle checkpoints” monitor the progression through the cell cycle and, by sensing possible defects affecting the crucial
steps of DNA replication and chromosome segregation, halt the progression of the cell cycle until all defects have been
repaired, and allow the transition to the next phase only in case all processes are properly completed (Barnum and O’Connell,
2014; Fig. 1). Also, in case the damages are too severe and cannot be fully repaired, the same control mechanisms induce a
permanent cell cycle arrest (cell senescence) or trigger programmed cell death (apoptosis). The concept of “cell cycle check-
point” was first introduced in 1988 by Hartwell and Weinert who observed that budding yeast harboring mutated RAD9 (a
gene encoding for a protein that halts the progression of the cell cycle in case of damaged DNA), in contrast to wild type yeast,
do not stop cell division to repair the irradiation-induced damages to the DNA and die because undergoing cell division with
damaged DNA (Weinert and Hartwell, 1988).
Lack of nutrients and of growth factors or the activation of inhibitory signaling pathways may block proliferating cells in early-
or mid-G1-phase. Otherwise, cells are committed to divide. However, errors in DNA replication or the exposure to both intrinsic
and extrinsic stressors, such as metabolism intermediates, carcinogens and ionizing radiation, may directly damage the DNA or
cause defective DNA replication or chromosome segregation. All these defects can be sensed by dedicated control mechanisms that
activate cell signaling pathways leading, on one hand, to the delay in G1, S and G2 phases or to prolonged cell cycle arrest in G1 or
G2 and, on the other hand, to the activation of DNA repair mechanisms. The signaling pathways that lead to the transient or
prolonged cell cycle arrest are initiated by two checkpoint kinases known as ATM (ataxia telangiectasia mutated) and ATR (AMT-
and Rad3-related), which sense double strand DNA brakes (DDB) and stretched single strand DNA brakes (SSDNA), respectively
(Abraham, 2001). ATM is physiologically present as an inactive homodimeric protein. The presence of DSB determines structural
chromatin changes even at long distance from the DBS that can be sensed by inactive ATM. Following the interaction with
chromatin, ATM homodimer dissociates with the consequent activation of ATM (Bakkenist and Kastan, 2003). Active ATM is then
recruited at the exact sites of DBS by molecular mediators such as NBS1 (Nijmegen breakage syndrome 1), BRCA1 (breast cancer 1),
and SMC1 (structural maintenance of chromosome)1 (Kitagawa et al., 2004; Uziel et al., 2003; Carson et al., 2003). In case of
stresses such as nucleotide depletion, that cause replication-fork arrest, and thus the presence of SSDNA, SSDNA are bound by the
replication protein A (RPA) that in turn recruits ATR (Cortez et al., 2001; Zou and Elledge, 2003) that is thus activated at the site of
replication-fork arrest (Osborn et al., 2002).
The signaling pathways initiated by ATM and ATR rely on two other groups of molecules, i.e., the checkpoint mediators or
adaptors and the checkpoint transducer Ser/Thr kinases or effector kinases, CHK1 and CHK2 (Kastan and Lim, 2000; Shiloh, 2003;
Petrini and Stracker, 2003). The checkpoints mediators promote the proper and fast localization and activity of ATM/ATR and
include the above mentioned NBS1, BCRA1 and the protein MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53
binding protein)1, for ATM, and the RSR and 9-1-1 complexes and claspin for ATR. The effector kinases CHK1 and CHK2 are the
6 Cell Cycle

main substrates of ATM and ATR, respectively, and, together with ATM and ATR, phosphorylate a broad range of molecules that are
involved in DNA repair, in the control of the cell cycle and in cell apoptosis (Bartek et al., 2004). Depending on the cell cycle phase
and the specific cell cycle checkpoint, ATM/ATR activate different signaling pathways as following described.

G1 and G1/S Phases Checkpoints

The G1 and G1/S checkpoints prevent cells harboring damaged DNA from entering the S-phase, even in case cells have already
passed the G1 restriction point. In G1-phase, the central player of the signaling pathway activated by ATM/ATR and CHK1/CHK2 is
the transcription factor and tumor suppressor p53. Indeed ATM/ATR and CHK1/CHK2 promote p53 stabilization (Craig et al.,
2003; Wahl and Carr, 2001; Kastan and Lim, 2000) by either directly phosphorylating it or by inactivating the ubiquitin ligase
mdm (mouse double minute)2 (Maya et al., 2001), responsible for p53 ubiquitin-dependent degradation. Among the genes
whose expression is regulated by p53, the CDK inhibitor p21 is fundamental in the G1 checkpoint (Abbas and Dutta, 2009).
Indeed, p21 inhibits the CDK2-cyclin E complex with the consequent arrest of the progression in G1 obtained by both hampering
the activation of molecules that promote the progression towards the S-phase and preserving active RB family proteins. The
mediated cellular response is quite slow since it is gene expression-based, but it can last for long time. This is the reason why the
activation of p53 may lead to a long-lasting effect and cause a prolonged arrest in G1. Also, ATM activates the p38 MAPK, in turn
promoting the expression of p16 that, by blocking CDK4/CDK6, causes G1 arrest (Kastan and Bartek, 2004). At later G1-phase
stages, in addition to the activation of p53, CHK1 and CHK2 also induce a faster even if short-lasting response that is based on the
inactivation of the CDK2-cyclin E inhibitor cdc25A (Donzelli and Draetta, 2003). Indeed in late G1, the increase in the expression
levels of cyclin E and cyclin A is paralleled by increased cdc25A, ATR and CHK1 levels. By phosphorylating cdc25A, ATR and CHK1
promote the ubiquitin-dependent degradation of cdc25A with the consequent inhibition of the CDK2-cyclin E complex.
The degree of DNA damage influences the levels of active ATM and ATR, in turn determining the levels of active p53 and p21, and of
p38 MAPK and p16. Depending on the levels of these molecules, cell fate is decided. High levels of p53 and p16 lead to a permanent
inhibition of the molecules that promote cell cycle progression dealing with cell senescence (Serrano et al., 1997). Furthermore, when
the levels of p53 are high enough to overcome a certain threshold, p53 triggers apoptosis (Fridman and Lowe, 2003).

S-Phase Checkpoint
DNA replication fires at specific sites known as replication origins. Different molecules localize at these sites, including the protein
CDC45 which serves to recruit the DNA polymerase a, in turn assembled into pre-replication complexes. The phosphorylation of
the molecules residing at the replication origin, which is required for replication firing, is mediated by CDKs and by the Dbf4-
dependent protein kinase (DDK) Cdc7 and determines both replication firing and the degradation of the same molecules so that
the same origin cannot fire again (Costanzo et al., 2003). CHK1 and CHK2, by blocking the activity of CDK2-cyclin A via cdc25B
inhibition, as described above for cdc25A, prevent CDC45 activation and loading on the chromatin, hampering DNA polymerase
a recruitment and blocking replication firing (Bartek et al., 2004; Falck et al., 2002). Also, other pathways involving other proteins
such as NBS1 take part in the regulation of the intra S-phase checkpoint (Yazdi et al., 2002; Taniguchi et al., 2002).

G2-Phase Checkpoint
The G2-phase checkpoint, also known as G2/M-phase checkpoint, has the function of preventing cells with damaged DNA, lasting
from the G1 and S phases or generated in G2, from undergoing mitosis. The mechanisms acting during the G2-phase checkpoint
converge on the inhibition of the mitotic complex CDK1-cyclin B. Different mechanisms may lead to CDK1-cyclin B inhibition,
and mainly rely on the inhibition of cdc25 family members by either degradation, mediated by ATM/ATR and CHK1/CHK2, or
sequestration, as a consequence of p38 MAPK signaling pathway activation (Mailand et al., 2002; Bulavin et al., 2001). As for cell
cycle arrest in G1, cells may undergo both a transient or a prolonged arrest in G2. Prolonged G2 arrest is mainly mediated by the
p53-p21 pathway (Taylor and Stark, 2001).
In addition to the described mechanisms, other checkpoint molecules that constitute the so called spindle checkpoint may be
activated during the M-phase. The spindle checkpoint senses defects in the attachment of the chromosomes to the spindle and
delays the progression through the M-phase to avoid the unequal segregation of chromosomes (London and Biggins, 2014;
May and Hardwick, 2006).

Cell Cycle Deregulation in Cancer

Uncontrolled cell proliferation due to constitutive mitogenic signaling or to lack of/defective responses to anti-mitogenic stimuli,
genome instability (GIN) and chromosomal instability (CIN) are hallmarks of human cancer (Hanahan and Weinberg, 2011) and
are associated with cell cycle deregulation. Activating mutations affecting CDKs, cyclins, and CDK-activating enzymes, inactivating
mutations affecting tumor suppressor genes, CDK inhibitors and checkpoint regulators promote unscheduled cell proliferation,
GIN and CIN (Malumbres and Barbacid, 2009; Kops et al., 2005). Cells harboring these genetic alterations show a proliferative
 Cell Cycle 7

advantage when compared to non-mutated cells and are prone to accumulate additional mutations that may lead to the acqui-
sition of more malignant phenotypes and thus promote tumor progression.
The vast majority of tumors shows alterations in the CDK4/CDK6-cyclin D-INK4-RB axis with merely all human tumors
showing genetic or epigenetic alterations in at least one component of this axis. CDK4 gene amplification or overexpression
have been observed in several malignancies including breast cancer (Samady et al., 2004; Takano et al., 1999; An et al.,
1999), melanoma, osteosarcoma and glioblastoma and it has been demonstrated that is it necessary to maintain breast
tumorigenesis (Yu et al., 2006). Also, increased CDK4 activity results from cyclin D1 overexpression which has been
reported in 60% of breast cancer, 40% of squamous carcinoma of head and neck and colorectal cancers and in 20% of
prostate cancers (Cheung et al., 2001; Gao et al., 2004; Hoechtlen-Vollmar et al., 2000; Reissmann et al., 1999). Finally, the
aberrant activation of BRAF or NRAS signaling, typical of melanomas as an example, translates into the activation of CDK4
(Yu et al., 2003). CDK6 gene amplification and overexpression has been reported in lymphomas, leukemias and gliomas,
among the others (Corcoran et al., 1999; Mendrzyk et al., 2005). Among INK4 genes, p16 is the most frequently altered
gene by inactivating point mutations, deletions or gene-silencing due to promoter methylation (Zhao et al., 2016; Serra and
Chetty, 2018). p16 is one of the most commonly altered tumor suppressor genes in human cancer with a particular high
frequency in melanoma, glioblastoma, lymphoid tumors, mesothelioma, nasopharyngeal, and pancreatic tumors (Serra
and Chetty, 2018 and references therein). The loss of p16 determines cell escape from senescence and the sustained activity
of CDK4/CDK6 (LaPak and Burd, 2014). RB1/p105, which is the most relevant target of CDK4/CDK6 and CDK2, is
frequently altered in cancer and typically in retinoblastoma and small cells lung carcinomas where it is lost in 80% of the
cases (Sherr and McCormick, 2002 and references therein). Also, RB1/p105 is the target of different proteins derived from
oncovirus, such as human papilloma virus (HPV) E7, adenovirus E1A and simian virus (SV) 40 large T antigen that, by
interacting with RB, determine its inactivation (Dyson et al., 1989; Giordano et al., 1989). Also, the deregulation of the
other two RB family members, RBL1/p107 and RBL2/p130, is frequent in human cancer (Dannenberg et al., 2004; Caputi
et al., 2002; D’andrilli et al., 2004; Claudio et al., 2002; Russo et al., 2005). Concerning CDK2, there are no mutations in
human cancers reported so far, however CDK2 abnormal activity can results from alterations affecting cyclins, CDK
inhibitors and related molecules (Santo et al., 2015). Also, cyclins E1 and E2 gene amplification is frequent in cancer and in
particular in uterine and ovarian cancers (Karst et al., 2014). CDK2 is also a downstream target of the oncosuppressor p53
as CDK2 is inhibited by the CDK inhibitor p21, a key p53-target gene, with p21-dependent CDK2 inhibition leading to cell
cycle restraining. Since p53 loss or mutations are extremely frequent in cancer, with the consequent deregulation of p21
(Abbas and Dutta, 2009), CDK2 activity is indirectly highly influenced by p53 status. Also, p21 loss or mutations are
frequent in cancer (Abbas and Dutta, 2009). In addition, p27, another important inhibitor of CDK2, is down-regulated in
several tumor entities with the consequent increased activity of cyclin E which can thus initiate the S-phase independently
from CDK4/CDK6 activity (Abukhdeir and Park, 2008). In contrast to interphase CDKs, a genetic deregulation of CDK1 is
not commonly linked to cancer. However, alterations affecting the p53, p21, p27 pathways as wells as the DNA damage
checkpoints may indirectly affect CDK1.
Increased CDK activity may also be consequent to mutations affecting CDK-activating enzymes and in particular the Cdc25
phosphatases whose deregulation or overexpression may cause unscheduled CDK activity (Galaktionov et al., 1995; Donzelli and
Draetta, 2003). In addition to genetic alterations directly affecting cdc25 phosphatases, cdc25 increased or persistent activity may
also result from an increase in the activity of the oncogene c-myc or of the signaling pathways downstream to ras oncogene
(Galaktionov et al., 1995, 1996).
Mutations affecting checkpoint proteins are highly frequent in cancer, with ATM-CHK2-p53 being the most commonly deregulated
checkpoint pathway in cancer (Falck et al., 2002). Mutations affecting ATM, ATR and CHK2 are associated with three syndromes, Ataxia
Telangiectasia, Seckel, and Li-Fraumeni, respectively, characterized by an increased susceptibility to cancer (Kastan and Bartek, 2004).
Also, CHK2 variants predispose to prostate and breast cancers (Kastan and Bartek, 2004). Mutations in the genes encoding for the
proteins BRCA1 and BCRA2, part of the S- and G2/M checkpoints, predispose to breast and ovarian cancers (Gabai-Kapara et al., 2014;
King et al., 2003; Venkitaraman, 2014; Walsh et al., 2006). p53 is the most frequently mutated gene in cancer (Kandoth et al., 2013). In
additions to mutations, p53 activity may also be abrogated upon binding to various viral oncoproteins such as SV40 T antigen and HPV
E6, or by mutations affecting the p53 regulator mdm2. A compromised activity of p53 translates into a reduction in the levels of the
CDK inhibitor p21 and thus into CDK hyperactivity (Muller and Vousden, 2013, 2014; Pflaum et al., 2014).
Given the well established role of deregulated CDKs and cyclins in tumorigenesis, their inhibition has been proposed as a
therapeutic anti-cancer strategy (Lapenna and Giordano, 2009; Sherr et al., 2016; Roskoski, 2016; Otto et al., 2017). Several
inhibitors have been generated so far and are currently in clinical trials. Three out of them, the CDK4/CDK6 inhibitors palbociclib,
abemaciclib and ribociclib have been recently approved for the treatment of advanced, hormone receptor-positive, post-
menopausal, breast cancer patients (Choo and Lee, 2018). However, the use of these inhibitors in other cancer patients did not
achieve the expected efficacy as anti-cancer therapy. CDK functional redundancy has been suggested as a potential explanation as
well as the lack of specificity for CDKs of many CDK inhibitors (Abate et al., 2013). This is the reason why the generation of novel
classes of CDK inhibitors is under investigation as well as the development of alternative therapeutic strategies aiming at inter-
fering with the cell cycle. Among the last ones, the use of microRNA (miRNA) targeting cell cycle regulators has been suggested.
Indeed, a class of miRNA targeting multiple components of the cell cycle regulating machinery has been identified and
demonstrated to be effective in counteracting cell proliferation in vitro, as well as tumor progression in vivo in tumor mice models
(Hydbring et al., 2017).
8 Cell Cycle

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Further Reading

Alberst, B., Johnson, A., Lewis, J., et al., 2016. Molecular Biology of the Cell, sixth ed. New York: Garland Science.
Enders, G.H., 2010. Cell Cycle Deregulation in Cancer. New York: Humana Press.
Morgan, D., Morgan, D.O., 2007. The Cell Cycle: Principles of Control. London: New Science Press.

Relevant Websites
https://cancer.sanger.ac.uk/cosmic
Catalogue of Somatic Mutations in Cancer.
https://www.cellsalive.com/cell_cycle.htm
The Cell Cycle
CELLS alive!.
https://www.nobelprize.org
The official website of the Nobel Prize.