REVIEW ARTICLES
ROLE OF CIRCADIAN CLOCK GENES IN THE
REGULATION OF CELL CYCLE PROCESSES
OLUWATOBI TEMITOPE SOMADE*
Department of Biochemistry, College of Natural Sciences,
Federal University of Agriculture Abeokuta, Ogun State, Nigeria
(Received, 28 April 2014)
One interesting branch of biochemistry which scientists are still trying to completely unravel is
the area of chronobiochemistry. The history of chronobiology is dated 400 B.C., when Androsthenes
observes that leaves of certain trees open during the day and close at night. It was only recently that
the first human clock gene was discovered. All living organisms possess an internal biological clock
referred to as the circadian (meaning about a day) clock that enables them to adapt to the continual
and daily cycle of day and night, as the earth evolves every 24 hours. This is initiated by a molecular
clock present in almost all cells in living things. The suprachiasmatic nuclei (SCN) in the
hypothalamus of the brain contain the master clock, and this master clock coordinates all other
peripheral clocks throughout the organism. Clock disruption by genetic or environmental factors has
been implicated in a number of pathological conditions. The circadian clock disorganization
predisposes humans and mice to cancer. Cell cycle and circadian cycle are two regulatory
mechanisms that indirectly or directly affect all biochemical reactions in cells. Therefore, the
disruption of one would result in the dysregulation of the other, with consequent adverse effects on
the cell. Lack of expression of clock genes has been found in a number of cancer types in humans.
Thus, internal (mutation) or external time cues (such as light/dark cycle and temperature) that may
disrupt the circardian rhythm may pose health risk in humans and other organisms.
Key words: Chronobiology, Circadian Clock, Cell Cycle, Suprachiasmatic nuclei.
INTRODUCTION
Chronobiochemistry which is concerned with the biochemical aspects of
chronobiology is the study of the inherent rhythmicity or periodicity (biological
clock) of living organisms and their activities. Circadian rhythm is the cyclical
change in the behavior and physiology of organisms with a periodicity of 24 hours
(1). This rhythm is elicited by a molecular clock that is located in almost all cells in
living organisms. All other peripheral clocks in the entire organism are controlled
by a master clock situated in the suprachiasmatic nuclei (SCN) in the anterior
*
Corresponding author (Email: [email protected]; Tel: +2348105030555)
ROM. J. BIOCHEM., 51, 2, 151–178 (2014)
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hypothalamus (Fig. 1). Disruption of the biological clock by environmental or
genetic factors has been implicated in numerous pathologic conditions (2–9).
Lately, animal studies suggested that circadian clock dysfunctions or deregulations
predispose humans and mice to cancer (5, 6).
Fig. 1 – The mammalian circadian timing system is a hierarchy of dispersed oscillators.
Source: Reppert and Weaver, 2002 (1)
THE ORGANIZATION OF THE SCN
The SCN which cannot be viewed as a homogeneous structure is composed
of different neuronal elements, each probably with its own specific function. The
neurons which are the components of these elements have intensive
interconnections and interactions, and have been amply demonstrated by
ultrastructural and immunochemical studies (10, 11). Certain specialization exists
within the SCN. The SCN consists of different neurons containing different
neuropeptides, such as vasopressin (VP), vasoactive intestinal peptide (VIP),
gastrin-releasing peptide or somatostatin. It has been demonstrated that several of
these neurons co-localize gamma amino butyric acid (GABA) or glutamate. About
30% of the SCN terminals contain GABA, as revealed through ultra-structural
studies (10, 12), and electrophysiological studies showed the presence of glutamate
as an additional SCN transmitter (13, 14). The combination of a large variety of
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peptides with or without amino acid transmitters within a single nucleus endows
the SCN with a rich variety of signaling properties. This set of SCN neurons with
their respective neurotransmitters serves to convey the circadian and light/dark
signal to hypothalamic target structures (15–17).
THE ANATOMICAL SETUP OF THE INTERNAL CLOCK
In higher organisms, the circadian pacemaker is situated in cells of specific
structures. Examples of such structures are certain regions of the brain (i.e., the
optic and cerebral lobes) in insects; the eyes in certain invertebrates and
vertebrates; and the pineal gland, which is located within the brain, in nonmammalian vertebrates. The circadian clock is located in two clusters of nerve
cells known as the suprachiasmatic nuclei (SCN) in mammals, which are located in
a region at the base of the brain called anterior hypothalamus (18).
In cells, maintenance of circadian periodicity depends on the nucleus.
Circadian rhythms are created by molecular oscillator mechanisms that are found
in virtually every cell of the human body. Oscillators in the cells, tissues and
organs are kept in step by a master clock located in the suprachiasmatic nuclei
(SCN) of the hypothalamus, which coordinates the tissue-specific peripheral
oscillators (Fig. 1) via humoral and neural mechanisms (18).
The molecular circadian oscillator in the SCN, and similarly in the peripheral
tissues, consists of a multiple gene mechanism of interacting positive and negative
feedback loops. The clock mechanism in humans and many animal species is
similar (1). The central master clock is kept entrained with the solar day-night
cycle (or artificial lighting regimen) through ganglion cells in the retina. Most
peripheral oscillators follow the circadian pacemaker with a 6 to 8 hours delay. The
coupling of peripheral oscillators to the circadian pacemaker may be altered or lost,
for example, by atypical times of restricted feeding schedules. The peripheral clock
in its timing can be affected by the intracellular metabolism, which is independent
of the SCN pacemaker.
The role of the SCN was demonstrated by the landmark discovery, in the
early 1970s, that by damaging the SCN in rats, the endocrine and behavioral
circadian rhythms could be disrupted and abolished (19). Also, if the SCN from
other animals is transplanted into those with an injured SCN, the circadian rhythms
could be restored. The function of the SCN as a master regulator of other rhythmic
systems was confirmed by similar research conducted using hamsters, which
revealed that restored rhythms exhibited the clock properties (i.e., the period or
phase of the rhythm) of the donor rather than those of the host (20). In mammals,
the discovery of SCN at the site of its primary regulation of circadian rhythmicity
provided researchers a focal point in their studies. Therefore, to understand day and
night timekeeping, the clock should be studied in the SCN.
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Recently, however, researchers were surprised to find out that circadian
rhythms could be maintained in cultured livers, lungs, and other tissues in vitro that
were not under SCN control (21). This observation indicates that most cells and
tissues of the body may be capable of modulating their activities based on the
circadian principles. However, this kind of finding cannot diminish or abolish the
major role played by SCN as the master circadian pacemaker that somehow
coordinates the entire daily temporal coordination of cells, tissues and the whole
organism at large. The physiological mode of action underlying this coordination
includes signals emitted by SCN that act on other nerve cells (i.e., neural signals)
or which are also distributed through the blood to other organs. The characteristics
of the circadian signal itself, or the specific manner in which SCN “communicates”
to the rest of the body remain unknown (22).
Although the effects of SCN lesions on numerous rhythms have been
mentioned, their outcomes on sleep are not totally clear. However, it is known that
lesions to the SCN clearly disorganize the consolidation and pattern of sleep in rats,
but have only minimal effects on the animals’ level of sleep or sleep need (23).
Due to this and some other reasons, it has been postulated by researchers that sleep
is subject to two essentially independent control mechanisms: [1] the circadian
clock that modulates the propensity for sleep, and [2] a homeostatic control that
reflects the duration of prior waking (i.e., “sleep debt”). However, in studies
conducted in squirrel monkeys it was observed that SCN lesions affect the amount
of sleep. Sleep studies in mice carrying mutational changes in two of the genes
influencing circadian cycles – i.e., the D-element binding protein (DBP) and Clock
genes – indicated that these mutations resulted in changes in sleep regulation (24, 25).
Many clock cells are located in the master clock in the SCN. Light
information is received by SCN directly to the retinohypothalamic tract (RHT),
which then entrains the clock to the 24-hour day. The entrained SCN coordinates
the timing of peripheral oscillators in other brain areas and all the peripheral
organs.
Component genes of the mammalian circadian clock
The genes responsible for running the internal clocks include: Circadian
locomotor output circuit kaput (clock), Period (per1, per2, per 3), Caseine kinase 1
epsilon (CK1ε), Cryptochrome (cry1, cry2), brain and muscle aryl hydrocarbon
receptor nuclear translocator (bmal1), cycle (cyc), white collar (wc1 and wc2),
frequency (freq) and others. These genes that control the circadian clock have been
found in people, mice, fish, plant, fruit flies, molds and cyanobacteria (18).
Cyanobacterium could be a model system for molecular approaches to the
circadian clock, because it is the simplest organism known to possess this activity.
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CIRCADIAN CLOCK IN NON-MAMMALIAN MODELS
Circadian clock in Cyanobacteria
Following unsuccessful results in finding time-dependent fluctuations in
bacteria, the circadian clock was firstly believed to exist only in eukaryotes. However,
circadian rhythms have been recently reported in several strains of nitrogen-fixing
cyanobacteria. Alternation of day and night dominates the metabolism of
photoautotrophic cyanobacteria that fix nitrogen (26). Photosynthesis by bacteria
during daylight generates oxygen from water. Since the enzyme nitrogenase is
sensitive to oxygen, nitrogen fixation has to be isolated from oxygen in
cyanobacteria (26). This problem is solved in filamentous cyanobacteria through
the development of specialized cells known as heterocysts. Vegetative cells form
oxygen through full-chain photosynthesis, but do not fix nitrogen, while
heterocysts turn off photosystem II in these multicellular species when they express
the enzyme nitrogenase (26). However, this separate segregation is not possible in
unicellular species. A temporal segregation of the two incompatible processes, i.e.
photosynthesis in the day and nitrogen fixation during the night, has been observed.
The first to describe a daily oscillation of nitrogenase activity of a marine
cyanobacterium were Mitsui et al. (27). Circadian rhythms of nitrogenase activity
and amino acid uptake in a freshwater nitrogen fixing strain, Synechococcus sp.
RF1, were later reported. They showed that the period of these rhythms was
temperature compensated, and the phases were reset by light signals (28–30).
Sweeney et al. (31) have also reported the circadian rhythm in cyanobacterial cell
division and the rhythms of nitrogen fixation and/or photosynthesis observed in
two cyanobacteria: Trichodesmium (32) and Cyanothece (33).
Bioluminescence
Bioluminescence provides a progressive method to monitor circadian rhythm,
as reported by numerous studies with the bioluminescent dinoflagellate, Gonyaulax
(34). The light output can be assessed and measured with great sensitivity without
damaging the cells. Therefore, designing a bioluminescence system that reports a
status of the oscillator could facilitate rhythm analyses (26). If the activity of a
particular gene promoter were controlled by an endogenous circadian clock, the
insertion of luciferase DNA downstream of such a promoter should be expressed
under clock control. By continuously providing the luciferase substrate, rhythmic
regulation of the circadian clock over the target promoter should be monitored as
daily oscillations in the intensity of bioluminescence (26). This strategy has been
successfully applied also in Arabidopsis (35) and Drosophila (36).
Understanding the circadian clock in Cyanobacterium
In understanding the circadian rhythm in cyanobacteria, a strain of
cyanobacterium, Synechococcus sp. PCC 7942, that is easily transformed by
exogenous DNA and is widely used in molecular genetic studies (37), is used as an
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example. The organism’s genome is 2.7 Mb in size, smaller than that of E. coli.
Synechococcus cells were transformed with a construct containing the promoter of
the psbA gene that encodes a major component of the photosystem II reaction
center (D1 protein) (38) driving the expression of the bacterial luciferase (luxAB)
gene (Fig. 2). LuxAB seems to be a good choice, because it is a volatile,
hydrophobic substrate, n-decanal and diffuses directly into Synechococcus.
Bioluminescence of the transformed Synechococcus cells oscillates for more than
10 cycles in constant light conditions, with a period of approximately 24 h (26).
Moreover, the phase of the rhythm is reset by a single dark pulse in a phasedependent manner and the period length is only slightly altered by temperature
(26). Thus, the rhythms of prokaryotic Synechococcus obey the three important
criteria for circadian rhythms that are established in eukaryotes (39). One of the
simplest model systems for the circadian oscillator is the transformed Synechococcus.
Fig. 2 – Circadian organization of Synechococcus; Source: Kondo and Ishiura, 2000 (26).
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This organization is made from an Oscillator, an Input and an Output, as
shown. Oscillator: In this prokaryotic model, kaiC expression is regulated by both
positive action through KaiA and negative feedback by KaiC. a, x, y and z
represent possible transcription factors, as yet unidentified. Input: External light
signals are transferred to the oscillator to reset the phase of the oscillation; the
relevant receptor molecule(s) and their action on the oscillator are unknown.
Output: Through unknown transcription factor(s), the oscillator controls a diverse
set of gene expression. A pathway that is influenced by rpoD2 and cpmA regulates
psbAI and other genes. Reporter: The PpsbAI:luxAB construct is inserted into a NS
(neutral site in the genome (39)) and, experimentally, serves as an artificial hand of
the clock. By administrating the luciferase substrate (decanal), the reporter strain
bioluminesces with a circadian rhythm, which is altered, often dramatically, by
induced mutations, most of which are in the kai gene cluster (26).
A central component of the cyanobacterial clock is the kai gene cluster
In Synechococcus, by introduction of a wild-type genomic DNA library into
an array of cells, efficient genetic complementation of mutant phenotypes is
possible (26). Therefore, a gene cluster made up of three novel open reading
frames was identified in fragments of DNA recovered from rescued clones of
several different rhythm mutants (40). Following this, more than 25 mutations –
including short-period, long period, low amplitude, and arrhythmic phenotypes –
were mapped to these three ORFs (26). This gene cluster was named kai, with the
ORFs termed kaiA, kaiB, and kaiC (Fig. 2) (40). Interestingly, the amino acid
sequences of Kai proteins do not share homology to any clock genes so far
identified in other organisms (41). Kai gene cluster deletion or inactivation of each
kai gene individually completely destroyed rhythmicity, and the replacement of the
whole kai gene cluster restored the rhythm (40). As a result, it is obvious that in
Synechococcus, all three kai genes are very important for the circadian rhythm.
Moreover, more than 50 clock mutants examined so far were all
complemented by the introduction of DNA from the kai gene cluster, regardless of
the diverse mutant phenotypes (26). These results suggest that the role of kai genes
depicts the main feature of the cyanobacterial circadian clock (26). In addition, the
normal growth of a kai knockout clone suggests that kai genes are dedicated to the
generation of circadian oscillations (26). Presently, a mutation in the pex gene,
which prolongs the circadian period by an hour, is the only rhythm-related gene
seen in Synechococcus (42).
Neurospora
In the fungus Neurospora crassa, circadian rhythm mutants were first
identified in the 1970s. Mutations in frq, wc-1, and wc-2 genes, caused period
length defects or arrhythmicity (43). The message levels of frq cycle, peak at
around circadian time (CT) 4 (44), whereas FRQ protein levels peak around CT 8,
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revealing that there is post-transcriptional delay (45). Frq mRNA level is
negatively regulated by FRQ protein (44), an effect assumed to occur at the
transcriptional level.
Drosophila
In Drosophila, the molecular-genetic study of circadian rhythms was initiated
with the discovery and eventual cloning of the period (dper) mutant gene based on
its perturbation of fly eclosion (i.e., the emergence of adults from their pupal cases)
and activity rhythms (46, 47). Timeless (dtim) was the next fly clock gene cloned,
identified both by the ability of dTIM to bind to the dPER protein and by positional
cloning of a mutant gene responsible for altered activity rhythms (48, 49). Both
dper and dtim mRNA level cycle peak in the early night around CT 14 (50, 51).
Fig. 3 – Scheme of the models for circadian oscillations in (A) Drosophila and (B) Neurospora.
Source: Gonze et al., 2000 (52).
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In fly circadian rhythm, per and tim genes expressions are negatively
regulated by the PER-TIM protein complex, and also TIM degradation is under the
influence of light. In Neurospora clock model, the transcription of the frq gene is
negatively controlled by the FRQ protein and also the gene expression is facilitated
by light. Looking at the two organisms, their models involve the transcription of
genes, and transport of protein in and out of the nucleus. There is accumulation of
tim, per, and frq mRNAs in the cytosol, accompanied by protein synthesis. Lastly,
in the two models, gene expression is regulated by nuclear forms of PER-TIM
complex and FRQ in Drosophila and Neurospora, respectively (52).
Zebrafish
Amongst vertebrates, Zebrafish is a strong model organism with unique
features for examining the mechanisms of the circadian clock and its entrainment
by light (53). The Zebrafish model proffers an abundance of molecular-genetic
techniques and bioinformatics tools, including methods for transgenesis, gene
knockdown, mutagenesis, and targeted genome modifications, combined with
advanced genomic annotation (54). When it comes to circadian biology, another
advantage of the Zebrafish, especially for studying the function of melatonin in the
regulation of circadian rhythms, is that this species is diurnal, just like humans
(54). The function of the pineal gland and the effects of melatonin on various
developmental, physiological and behavioural processes have been investigateded
in Zebrafish by numerous researchers (55–59). Some of their findings have shown
that exogenous melatonin administration results in reduced locomotor activity and
exacerbates a sleep-like state (60–62) and functions to schedule the timing of
reproduction (55) and feeding (58).
Mechanisms of light-induced clock gene expression in Zebrafish
Regulation of the light-induced Zebrafish clock gene, per2, has been
investigated in order to explore the mechanisms underlying synchronization by
light (54). Regulation of the Zebrafish per2 promoter was first explored in vivo,
resulting in the identification of a minimal promoter fragment that is adequate to
drive per2 expression and, mostly, regulation by light (63). The existence of a
photoentrainable clock system within Zebrafish cells has immensely promoted the
unraveling of the regulatory mechanism surrounding the light-induced per2
expression (54). These ex vivo studies in Zebrafish Pac-2 cells showed a novel
molecular mechanism that concomitantly propels clock- and light-regulated
transcription (63). This mechanism is mediated by closely spaced E-box and D-box
regulatory elements that are located in the proximity of the per2 transcription start
site (63). Light-induced transcriptional activation was shown to be mediated by the
D-box element and a D-box binding transcription factor, tef-1 (63). Eleven
additional Zebrafish D-box-binding factors from the PAR and E4BP4 family have
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since been cloned and characterized. The expression of nine of these factors is
stimulated in the pineal gland and coordinated to varying extents by the clock
and/or by light (64). Moreover, it was shown that the expression of some of these
factors possesses a somewhat similar clock- or light-driven regulation in Zebrafish
Pac-2 cells (65). A systematic functional analysis of the cry1a promoter showed
that a single D-box directs light-induced expression of this clock gene and that
PAR factors are able to transactivate expression from this D-box element (65). The
D-box-mediated pathway is also involved in the regulation of other light-induced
genes (66, 67). Thus, D-box enhancers appear to act as important elements in lightdriven signaling in both the pineal gland and the cell lines, heading towards a
somewhat similar mechanism of light-entrainment in the central and peripheral
clocks. Interestingly, this is different from the situation in the mammalian circadian
timing system, where D-boxes appear to function as regulatory elements of clock
output pathways (68).
With the aim of further exploring the mechanisms by which the central
circadian clock is conjoined by light, both RNA-seq and microarray technologies to
characterize the light-induced coding transcriptome of the Zebrafish pineal gland
were used (69), resulting in the identification of multiple light-induced mRNAs.
An interesting outcome of this approach was the identification of 14-core clock and
clock accessory loop genes as light-induced genes in the pineal gland, including
per2 and cry1a, most of which are members of the negative limbs of the molecular
oscillator (54) (Fig. 4).
Fig. 4 – Model of the regulation of the molecular clockwork in the zebrafish pineal gland by light.
Source: Ben-Moshe et al., 2014 (54).
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Exo-rhodopsin served as a receiver of light at the cell membrane to the
nucleus by signal transduction pathways. This led to the up-regulation and
expression of negative elements in the clock-work circuitry. The microRNAs
(miRNAs) induced by light caused the inhibition of clock and clock-controlled
genes. Arrow ends depict activation; lines with flat end show inhibition.
MOLECULAR GENETICS OF CIRCADIAN RHYTHMS
Mutations have been introduced into the DNAs of Drosophila melanogaster
and Neurospora. The mutation resulting from these organisms then were screened
for rhythm abnormalities (18). This mutagenesis approach led to the identification
of the first circadian clock mutants, which were called period (per) and frequency
(frq, pronounced “freak”). The genes that carried the mutations in these organisms
were cloned in the 1980s (70). However, researchers found it difficult to isolate the
equivalent genes in mammals. In early 1990s, scientists began a similar
mutagenesis screening approach in the mouse and described the first mouse
circadian mutation, called clock, in 1994 (71). The gene affected as a result of this
mutation was the first mammalian circadian clock gene to be cloned in 1997 (71).
Similar to the mutants of the per and frq genes, the mutation to the clock gene both
affected the free-running rhythm period (i.e., lengthened the period) and resulted in
a loss of persistence of circadian rhythms under constant and normal environmental
factors. Both the clock and per mutant in mice and flies respectively were the first
to be identified using such a mutagenesis approach in which the mutation
manifested as altered behavior rather than an altered physiological process (18).
Since the clock gene was discovered in mice, the number of genes of the
circadian clock identified in mammals has increased in a remarkably short period
of time. Researchers have identified three mammalian genes that are similar to per
gene in both their structure (i.e., nucleotide sequence) and function (71, 72). Some
of the proposed circadian clock genes have been identified mostly based on their
similarity in sequence to clock genes of Drosophila and examination of the
corresponding mutants’ behaviour did not show that they might have clock
properties. The findings thus far have clearly indicated the outline of a pacemaker
that is based on a feedback cycle of gene expression.
THE MAMMALIAN CIRCADIAN CLOCK MECHANISM
The cellular mechanism of circadian rhythmicity involves the regulation of
three Period genes (Per 1–3) and two Cryptochrome genes (Cry1 and 2) (73)
(Fig. 5). Currently it is thought that transcription of Per and Cry genes is driven by
accumulating clock:bmall complexes, followed by its binding to E-box elements
(74–77). Later on, per2 and cry2 proteins complexes translocate to the nucleus,
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where they block transcription mediated by clock. Concomitantly, bmal1 mRNA
levels are up-regulated by per2 leading to the formation of clock:bmal1 complexes,
which stimulate the transcription of per2 and cry2 and restart the cycle (78, 79).
Fig. 5 – Mammalian circadian clock mechanism. Source: Reppert and Weaver, 2002 (1).
INTERACTION BETWEEN CIRCADIAN CYCLE AND CELL CYCLE
Two regulatory mechanisms that indirectly or directly affect all biochemical
reactions in cells are the cell cycle and the circadian cycle (Fig. 6). Therefore,
dysfunction of one would cause disorganization of the other, with fatal
consequences on the cell (80–82). It has been documented that in proliferating
cells, major circadian clock components affect the cell cycle by controlling wee1
expression, a kinase that regulates Cdc2 activities and hence the transition from
G2- to M-phase of cell cycle (83). In another study it was concluded that mice with
per2 mutation possessed constitutively high levels of c-Myc expression, a cell
growth/proliferation gene, and lowered the p53 gene expression, which plays a
major role in the cell cycle G1-S checkpoint (84).
The cell cycle and circadian clock are globally two regulatory systems at the
cellular and organismic levels and hence, one would expect them to interface
(Fig. 6). As a matter of fact, the systems interface at some points that are critical.
The anti-mitotic kinase wee1 is regulated positively by Bmal1-Clock heterodimers
(83, 85, 86), and also the transcription of c-Myc is repressed by Bmal1-Clock or
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Fig. 6 – Mechanisms employed by core clock genes in the control of cell cycle progression.
Source: Borgs et al., 2009 (90).
(A) RORα and REV-ERBα bind the same RORE element presents in p21 promoter. Transcription of
p21 is activated by RORα, which results in the inhibition of CDK2/cyclin E and inhibits G1/S progression.
p21 transcription is inhibited by REV-ERBα, resulting into the activation of CDK2/cyclin E complex
and G1/S progression. (B) c-myc transcription is inhibited by PERs, causing its inability to activate
cyclin D1. G1/S progression is thus indirectly inhibited by PERs. (C) BMAL1/CLOCK or
BMAL1/NPAS2 heterodimers and CRYs recognize E-boxes located in Wee1 promoter, thus, activate
or inhibit Wee1 transcription, respectively. CDK1/cyclin B complex is inhibited by WEE1 and thus
G2/M progression is repressed. (D) PER1 and TIM play the role of co-factors, activating ATM or
ATR, which then phosphorylate Chk2 or Chk1, causing cell cycle arrest (90).
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Bmal1-NPas2 (84). In per2 mutant mice, there was a highly elevated c-Myc
transcription. The reason is that Bmal1 transcription is positively regulated by per2,
and so, in the absence of per2, Bmal1 which is known to repress c-Myc is down
(84). Infrared-induced lymphoma in these mutant mice was attributed to the
elevated c-Myc (84). Apart from the cell/circadian cycle-connection at the
transcriptional level, the two processes also interface at the protein–protein
interaction and signal modulation level. Firstly, the protein needed for a normal
circadian rhythm in mice, known as Timeless protein (87), directly associates with
the checkpoint proteins ATR and Chk1 of the cell cycle in such a way that downregulation of the circadian Timeless protein interferes with the branch of Chk1ATR3 of DNA damage checkpoint response (88). Secondly, it has been reported
that per1 associates with both Chk2 and ATM proteins and directly takes part in
the signaling of ATM3-Chk2 DNA damage pathway (89). It has also been reported
that the overexpression of per1 leads to cell death in numerous cancerous cell lines,
presumably through the activation of the ATM3-Chk2 signaling pathway, and
halting proliferation of cell and stimulating apoptosis (89).
The circadian nature of cell division
A fundamental property of circadian clocks in eukaryotes is that they are
based on cell-autonomous interacting transcriptional/translational feedback loops
that propel cyclical gene expression, which ultimately underlies many of the
rhythms displayed by organisms (91). Numerous cell cycle oscillator core genes
also show a circadian expression (92). Adjustment in expression levels of various
canonical circadian clock genes leads to changes in the level of several cell cycle
genes, as well as in numerous cell cycle disorders (90).
The gating attribute of the circadian clock may be conserved from unicellular
organisms to vertebrates. In cyanobacteria, Yang et al. (93) showed a reduction in
the progress of cell cycle in a specific phase of the circadian interval. The gene per
is one of the genuine circadian clock canonical genes (94). The mammalian period
paralogues Per1 and Per2 are molecularly connected to repression of G1-S
transition, while the circadian transcription factors Bmal and Clock are linked to
G2-M transition (90, 83, 95, 96). An additional important role for BMAL1 protein
is in blocking cells from undergoing S-phase, while reactive oxygen species (ROS)
levels are raised (97). Further evidence for a molecular crosswalk mode of gating
operation has been recently found by Kowalska et al. (98), who showed that
NONO, a multifunctional nuclear protein, worked together with the circadian
protein PER in coordinating the circadian expression of the cell cycle checkpoint
gene p16-Ink4A. The circadian regulation of p16-Ink4A via NONO is important for
gating exit from cell cycle G1 phase (98, 99).
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Timeless (tim) is another implicating putative circadian clock gene. It is an
important element of the circadian oscillator in the fruit fly Drosophila
melanogaster (94). In mammals, the probable descendant homolougue TIM protein
is not a true orthologue of Drosophila TIM, and its functional link with the
circadian oscillator is under debate (94). A recent study proposed a probable role
for TIM in the mammalian circadian clock speed and resetting (100).
Looking at the circadian mode of cell proliferation and the causal association
with the DNA damage checkpoint, an important question is whether changing the
normal function of circadian cycle can also affect the dynamics of cell division,
with further comprehensible implications for tumor growth (101–104).
Circadian clock and cell cycle genes
The expression of about 2 to 10 percent of all mammalian genes is under
clock control. These are tissue- or organ-specific in the expression of their patterns,
although a few genes that are controlled by circadian clock, encoding cell cycle
progression, are expressed in more than one organ (6). The genes participating in
the circadian clock and those of the cell cycle/DNA damage checkpoints are
functionally or molecularly linked, which is crucial in maintaining the integrity and
stability of the genome (105). Cell proliferation key regulators such as wee1, in
normal tissues regeneration are directly regulated by the molecular machinery of
the circadian clock system (83).
p21 belongs to the Cip/Kip family of cyclin-dependent kinase inhibitors. It
regulates cell cycle progression negatively by inhibiting cyclin E-cdk2 complexes
activity during G1 phase progression of the cell cycle and inhibiting DNA
replication via binding to proliferating cell nuclear antigen (106) (Fig. 7). p21 is
activated by p53 after DNA damage and plays an important role during epidermis
differentiation (108,109,110,111). In Bmal1-null mice, a dramatic increase of p21
mRNA levels from Zeitgeber time 4 (ZT4) to Zeitgeber Time 16 (ZT 16) with 8 to
12-fold overexpression at ZT8 was observed, as compared with wild type animals.
This observation suggests that Bmal1 represses p21 transcription (106). Therefore,
p21 is considered as a clock-controlled gene (106). p53, key regulator of p21 has
also been shown to oscillate in the human oral mucosa (112, 113). In Bmal1 mutant
and wild type livers, p53 is expressed identically in which no significant
rhythmicity can be detected. p21 is regulated by clock-genes via p53 independent
pathways, and therefore it is not a putative circadian regulator of p21 (106). Also, a
known clock target Wee1, regulates the G2/M transition (83) (Fig. 7). It is
expressed at intermediate and constant levels in Bmal1 animals. Cell cycle
encoding genes, Cdk6 and Ccne2 that are involved in the G1 phase and G1/S
transition, respectively, showed no significant oscillation in wild type liver or
alteration of their expression in the mutant liver (106).
166
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16
Table 1
Molecular relationships between circadian genes and cell cycle (90)
Cell
cycle
phase
G1-S
Circadian
genes
Target
genes
Effect
Reverbα
RORα
Per1
p21
Inhibition of p21
Induction of G1-S transition (106)
p21
Activation of p21
Repression of G1-S transition (106)
Cyclin D1 Inhibition of cyclin D1 Repression of G1-S transition
(95)
Per2
c-myc
Inhibition of c-myc
Cell cycle arrest (96)
G2-M BMAL1/CLOCK Wee1 Activation of Wee1
Inhibition of cyclin B/CDK1
BMAL1/NPAS2
Repression of G2-M transition (83)
Cry1
Wee1
Inhibition of Wee1
Activation of cyclin B/CDK1
Induction G2-M transition (83)
CK1ε
Cyclin B1 Phosphorylation of cyclin B1, A2
Activation G2-M
CyclinA2
transition (107)
DNA
Per1
ATM
Activation of ATM
Induces phosphorylation of
Chk2
damage
Cell cycle arrest (8, 89)
Tim
ATR
Activation of ATR
Induces phosphorylation of Chk1
Cell cycle arrest (8, 88)
Fig. 7 – Molecular links between circadian clock and cell cycle genes.
Source: Grechez-Cassiau et al., 2008 (106).
Pathways showing how the cell division cycle is related to circadian clock signaling. This happens at
multiple levels via wee1, per1, and p21. Therefore, these links suggests that the circadian clock can
regulate the process of cell proliferation at different levels including the G1 phase, G2/M transition,
and the DNA damage response pathway (106)
17
Circadian clock-cell cycle relationships
167
Circadian cycle and cell cycle checkpoints
Current clock models presume that cry and per function as heterodimers. It
has been shown that in mutated per2 mice, c-Myc is up-regulated while p53 is
down-regulated, leading to a general cell cycle dysregulation (84). It is therefore
expected that the cry mutant fibroblasts would show some defects in the cell cycle
checkpoint and also the mutated cry mice, like the per2 mutants, would be prone to
cancer.
Circadian cycle genes and apoptosis
Research studies have shown that, when per2 is expressed at high level in
MCF-7 breast cancer cells, a significant growth-inhibitory effect was stimulated
(114). When per2 and cry2 were expressed together, a much more enhancement of
growth-inhibition was noticed when compared to per2 only expressing cells (114).
Cry2 expression alone had no significant effect on the proliferation of MCF-7 cell,
indicating that cry2 is independently not a critical regulator of cell cycle but that it
participates in conjunction with per2 to regulate cell cycle and cell death in breast
cancer cells (114).
Cell proliferation inhibition by per2 and per2/cry2 may be associated to
several events, like cell cycle alteration and induction of apoptosis (114). Per2
alone or per2/cry2 expression inhibited MCF-7 cell cycle at the G1/S border. The
anti-proliferative effects of per2 are attributed to the stimulation of apoptosis rather
than prolonging or halting the cell cycle, as morphological changes – e. g.,
rounding up and detachment that are consistent with programmed cell death – are
induced in per2 expressed MCF-7 cells (114). Apoptosis induced by per2 was
confirmed by a significant increase in polyADPribose polymerase (PARP)
cleavage, indicating the activation of caspases (115).
It has been reported that genes controlled by clock, such as c-Myc, p53, and
cyclins, are involved in the regulation of cell cycle and apoptosis (84). Cyclin D1 is
a major regulator of cell cycle, up-regulated by estrogen, which is an established
breast cancer mitogen (116). Therefore, the down-regulation of cyclin D1 is
important in cell cycle arrest. Cyclin D1 level is significantly down-regulated by
per2 expression in MCF-7 cells, which may contribute to the G1 arrest of the cell
cycle (114). Impaired cell cycle regulation, inhibition of apoptosis and genomic
instability are consequences of p53 loss in many cancers (114). In cells, the tumor
suppressor p53 can induce a transient arrest in G1, given cells opportunity to repair
damaged DNA (117).
The main way by which p53 mediates tumor suppression is through
elimination of abnormally proliferating cells (96, 117). Per2 expression in MCF-7
cells significantly raised p53 levels (114). In per2 expressing breast cancer cells
there is an elevation in p53 expression, which may contribute, at least in part, to
168
Oluwatobi Temitope Somade
18
both G1 arrest of cell cycle and apoptosis (114). Tumor cell apoptosis may be
induced by per2 via the p53-mediated mitochondrial signaling pathway (114).
The tumor suppressive property of per2 – as indicated by the induction of
apoptosis, inhibition of cell growth, reduced colony formation and growth in soft
agar – has been clearly shown (84, 114, 118). Although per2 can function as a
tumor suppressor independently, its activity is significantly enhanced in the
presence of cry2, its clock partner (114).
Table 2
Circadian clock gene disruption results in cell cycle disorders (90)
Trigger
SCN ablation
Per1 overexpression
Per2 downregulation
Per2 overexpression
CK1ε inhibition
Clock downregulation
Bmal1 downregulation
Cry downregulation
Effect on proliferation
Increases proliferation (119)
(normal or malignant)
Leads to cell growth inhibition (89,118)
Increases cyclin A, cyclin D1, mdm2, c-myc, βcatenin, cyclin E
Increases proliferation (84,120,121,122)
γ-irradiation on Per2Brdm1 mice (121)
downregulates p53
Increases proliferation
Increases p53 transcription (96,120,123)
Increases apoptosis
Inhibits tumor formation
Decreases β-catenin and cyclin D (124)
Decreases cell proliferation
Decreases cyclin B1, cyclin A2 (107)
Decreases cell proliferation
γ-irradiation on Clock mutant mice (125)
Accelerates premature aging but no effect
on proliferation
Bmal1 mutant mice with or without γirradiation (125,126)
Accelerates premature aging but no effect
on proliferation
Cry1-/- ; Cry2-/- double mutant mice (85)
no effect on proliferation
Circadian cycle genes and cancer
Going by the molecular characterization of the mammalian oscillator, it has
been clearly understood that the circadian clock takes part in cellular pathways that
are critical for cell division and tissue homeostasis (127–129). Through the
clock/bmal1 heterodimer, transcriptional circadian regulation goes beyond clock
genes to encompass different clock controlled genes, some of which are key cell
cycle regulators, such as c-Myc, cyclin D1, and wee1 (130, 131). The importance of
proper circadian cycle regulation to cell cycle progression and the DNA-damage
response has been recently shown by studies in mice with a mutation in cry and
19
Circadian clock-cell cycle relationships
169
per2 genes, causing improper cell division and therefore increased their sensitivity
to radiation (83, 84). Apoptosis, cell cycle arrest, tumor suppression, growth
inhibition, and loss of clonogenic ability were observed in the induction of per2
expression in cancer cells (132).
Independent studies revealed that mper2 overexpression induced apoptosis
only in cancer cells, but not also in normal NIH 3T3 cell lines (96). Per2 was
identified as one of C/EBPα’s target genes; a CCAAT/enhancer binding protein
(132) involved in the regulation of cell growth and could play a key role in acute
myeloid leukemia, lung and liver cancers (132). Per1 protein deregulation was
commonly seen in human endometrial and pancreatic cancer cells but not in the
adjacent normal cells (89, 133, 134). A study has shown that 95% of breast tumors
display dysregulated levels or lack of per1 and per2 in the tumor cells when
compared with adjacent normal cells (135). In lung cancer patients, 70% showed
significantly lower levels of per1 gene expression in tumor tissues compared with
matched normal tissues (136).
Light at night, chronodisruption and malignancy
Light is the most eminent environmental signal that coordinates daily and
seasonal timing and as such is a powerful regulator of physiology and behavior
(94). Modern life often faces disturbances in natural light/dark cycles. Light phase
is prolonged by light-at-night (LAN) and various professions involve night work or
shift work. In addition, lights turned on during the dark phase generate light pulses,
further stimulating interferences. The resulting chronodisruption can cause havoc
for circadian clock organization (94). A substantial inference is whether such
disruption of circadian organization and entrainment also affects the dynamics of
cell division. If so, the disorganization of the circadian oscillator per se can further
stimulate cancer growth and tumor progression (9, 102, 137).
There is increased evidence associating LAN to various malignancies in
general and to breast cancer in particular. Nighttime satellite images and electricity
consumption were monitored to study the correlation between LAN and various
cancer incidents. Results indicate a significant linkage between rates of breast
cancer and LAN (138, 139). Wu et al. (140) revealed that to some extent, exposing
transgenic rats to LAN accelerated tumor growth in vivo via continuous activation
of IGF-IR/PDK 1 signaling.
Nightshift work has long been linked with ill-health cancer risk (141–144). In
the past few years there have been numerous supportive meta-analyses and facts
showing that night work does play a role in breast cancer (145, 146).
Chronodisruption by dim light at night or chronic jet lag speeds up tumor growth
(147). The circadian oscillator also mediates daily cycles of detoxification and drug
metabolism, i.e. toxicity and anti-cancer xenobiotic efficacy is modified with the
circadian time (148). As a result, a corresponding approach can consider
chronotherapy by means of using the circadian oscillator to down-regulate cell
proliferation and malignant growth factors (149).
170
Oluwatobi Temitope Somade
20
CONCLUSION
Irregular or disruption of the circadian rhythm usually has a negative effect
(Fig. 8). Damage to clocks (Fig. 8) is believed to have adverse health effects on
peripheral organs, mainly in the development or exacerbation of cancer (5, 6, 84,
96, 114, 151), cardiovascular disease (151), diabetes mellitus type 2 (150) and
some other health problems.
Fig. 8 – General Overview of the Consequences of Clock Genes Disruption
Source: Wulff et al., 2010 (150).
21
Circadian clock-cell cycle relationships
171
Also, hormonal problems, such as defects in melatonin production, caused by
the disruption of the circadian rhythm, may promote the risk of cancer
development (152). Therefore, dysfunctional circadian system, lack of clock genes
expression, or gene mutations that may ultimately disturb cell cycle activities could
pose serious health risks in humans.
Competing interests: None.
REFERENCES
1. Reppert SM, Weaver DR, Coordination of circadian timing in mammals, Nature, 418, 935–941
(2002).
2. Schwartz WJ, A clinician’s primer on the circadian clock: Its localization, function, and resetting,
Adv. Intern. Med., 38, 81–106 (1993).
3. Sancar A, Cryptochrome: The second photoactive pigment in the eye and its role in circadian
photoreception, Ann. Rev. Biochem., 69, 31–67 (2000).
4. Hastings MH, Reddy AB, Maywood ES, A clockwork web: Circadian timing in brain and
periphery, in health and disease, Nat. Rev. Neurosci., 4, 649–661 (2003).
5. Stevens RG, Circadian disruption and breast cancer: From melatonin to clock genes,
Epidemiology, 16, 254–258 (2005).
6. Fu L, Lee CC, The circadian clock: Pacemaker and tumour suppressor, Nat. Rev. Cancer, 3, 350–361
(2003).
7. Kobayashi M, Wood PA, Hrushesky WJ, Circadian chemotherapy for gynecological and
genitourinary cancers, Chronobiol. Int., 19, 237–251 (2002).
8. Kondratov RV, Antoch MP, Circadian proteins in the regulation of cell cycle and genotoxic stress
responses, Trends Cell. Biol., 17, 311–317 (2007).
9. Levi F, Schibler U, Circadian rhythms: Mechanisms and therapeutic implications, Ann. Rev.
Pharmacol. Toxicol., 47, 593–628 (2007).
10. Buijs RM, Wortel J, Hou YX, Colocalization of gammaaminobutyric acid with vasopressin,
vasoactive intestinal peptide, and somatostatin in the rat suprachiasmatic nucleus, J. Comp.
Neurol., 35, 8343–352 (1995).
11. Romijn HJ, Sluiter AA, Pool CW, Wortel J, Buijs RM, Evidence from confocal fluorescence
microscopy for a dense, reciprocal innervation between AVP-, somatostatin-, VIP/PHI-, GRP-,
and VIP/PHI/GRP-immunoreactive neurons in the rat suprachiasmatic nucleus, Eur. J.
Neuro., 9, 2613–2623 (1997).
12. Castel M, Morris JF, Morphological heterogeneity of the GABAergic network in the
suprachiasmatic nucleus, the brain’s circadian pacemaker, J. Anat., 196, 1–13 (2000).
13. Hermes MLHJ, Coderre EM, Buijs RM, Renaud LP, GABA and glutamate mediate rapid
neurotransmission from suprachiasmatic nucleus to hypothalamic paraventricular nucleus in
rat, J. Physiol., 496, 749–757 (1996).
14. Cui LN, Coderre E, Renaud LP, Glutamate and GABA mediate suprachiasmatic nucleus inputs to
spinal-projecting paraventricular neurons, Amer. J. Physiol. Regul. Int. Comp. Physiol., 1,
1281–1283 (2002).
15. Kalsbeek A, Vanheerikhuize JJ, Wortel J, Buijs RM, A diurnal rhythm of stimulatory input to the
hypothalamo–pituitary–adrenal system as revealed by timed intrahypothalamic administration
of the vasopressin V-1 antagonist, J. Neuro., 16, 5555–5565 (1996b).
16. Kalsbeek A, Garidou ML, Palm IF, Van der Vliet J, Simonneaux V, Pevet P, Buijs RM,
Melatonin sees the light: blocking GABA-ergic transmission in the paraventricular nucleus
induces daytime secretion of melatonin, Eur. J. Neuros., 12, 3146–3154 (2000).
172
Oluwatobi Temitope Somade
22
17. Buijs RM, Kalsbeek A, Hypothalamic integration of central and peripheral clocks, Nat. Rev.
Neuros., 2, 521–526 (2001).
18. Vitaterna MH, Takahashi JS, Turek FW, Overview of circadian rhythms, J. Alch. Res. Health.,
25, 85–93 (2001).
19. Gillette MU, SCN electrophysiology in vitro: rhythmic activity and endogenous clock properties,
in: Suprachiasmatic Nucleus: The Mind's Clock, Klein DC, Moore RY, Reppert SM, eds,
Oxford University Press, New York, NY, 1991, pp 125-143.
20. Ralph MR, Foster RG, Davis FC, Menaker M, Transplanted suprachiasmatic nucleus determines
circadian period, Science, 24, 7975–978 (1990).
21. Yamazaki S, Numano R, Abe M, Hida A, Takahashi RI, Ueda M, Block GD, Sakaki Y, Menaker
M, Tei H, Resetting central and peripheral circadian oscillators in transgenic rats, Science,
288, 682–685 (2000).
22. Stokkan KA, Yamazaki S, Tei H, Sakaki Y, Menaker M, Entrainment of the circadian clock in
the liver by feeding, Science, 291, 490–493 (2001).
23. Mistlberger RE, Bergmann BM, Rechtschaffen A, Relationships among wake episode lengths,
contiguous sleep episode lengths, and electroencephalographic delta waves in rats with
suprachiasmatic nuclei lesions, Sleep, 10, 12–24 (1987).
24. Naylor E, Bergmann BM, Krauski K, Zee PC, Takahashi JS, Vitaterna MH, Turek FW, The
circadian clock mutation alters sleep homeostasis in the mouse, J. Neuro., 20, 8138– 8143
(2000).
25. Franken P, Lopez-molina L, Marcacci L, Schibler U, Tafti M, The transcription factor DBP
affects circadian sleep consolidation and rhythmic EEG activity, J. Neuro., 20, 617–625
(2000).
26. Kondo T, Ishiura M, The circadian clock of cyanobacteria, Bio Essays, 22, 10–15 (2000).
27. Mitsui A, Kumazawa S, Takahashi A, Ikemoto H, Cao S, Arai T, Strategy by which nitrogenfixing unicellular cyanobacteria grow photoautotrophically, Nature, 323, 720–722 (1986).
28. Huang TC, Tu J, Chow TJ, Chen TH, Circadian rhythm of the prokaryote Synechococcus sp.
RF-1, Plant Physiol., 92, 531–533 (1990).
29. Chen TH, Chen TL, Hung LM, Huang TC. Circadian rhythm in amino acid uptake by
Synechococcus RF-1, Plant Physiol., 97, 55–59 (1991).
30. Huang TC, Chen HM, Pen SY, Chen TH, Biological clock in the prokaryote Synechococcus RF-1,
Planta., 193, 131–136 (1994).
31. Sweeney BM, Borgese MB, A circadian rhythm in cell division in a prokaryote, the
cyanobacterium Synechococcus WH7803, J. Phycol., 25, 183–186 (1989).
32. Roenneberg T, Carpenter EJ, Daily rhythm of O2-evolution in the cyanobacterium Trichodesmium
thiebautii under natural and constant conditions, Marine Biol., 117, 693–697 (1993).
33. Schneegurt MA, Sherman DM, Nayar S, Sherman LA, Oscillating behavior of carbohydrate
granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC
51142, J. Bacteriol., 176, 1586–1597 (1994).
34. Morse DS, Fritz L, Hastings JW, What is the clock? Translational regulation of circadian
bioluminescence, Trends Biochem. Sci., 15, 262–265 (1990).
35. Somers DE, Webb AA, Pearson M, Kay SA, The short-period mutant, toc1-1, alters circadian
clock regulation of multiple outputs throughout development in Arabidopsis thaliana,
Development, 125, 485–494 (1998).
36. Stanewsky R, Kaneko M, Emery P, Beretta B, Wager-Smith K, Kay SA, Rosbash M, Hall JC, The
cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila, Cell, 95,
681–692 (1998).
37. Tsinoremas NF, Kutach AK, Strayer CA, Golden SS, Efficient gene transfer in Synechococcus sp.
strain PCC 7942 and PCC 6301 by interspecies conjugation and chromosomal recombination,
J. Bacteriol., 176, 6764–678 (1994).
38. Golden SS, Light-responsive gene expression and the biochemistry of the photosystem II reaction
center, in: The molecular biology of cyanobacteria, Bryant DA, ed, Dordrecht, The
Netherlands: Kluwer Academic, 1994, pp 693–714.
23
Circadian clock-cell cycle relationships
173
39. Kondo T, Strayer CA, Kulkarni RD, Taylor W, Ishiura M, Golden SS, Johnson CH, Circadian
rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in
cyanobacteria, Proc. Natl. Acad. Sci. USA, 90, 5672–5676 (1993).
40. Ishiura M, Kutsuna K, Aoki S, Iwasaki H, Andersson CR, Tanabe A, Golden SS, Johnson CH,
Kondo T, Expression of a gene cluster kaiABC as a circadian feedback process in
cyanobacteria, Science, 281, 1519–1523 (1998).
41. Dunlap J, Molecular basis for circadian clocks, Cell, 96, 271–290 (1999).
42. Kutsuna S, Kondo T, Aoki S, Ishiura M, A period-extender gene, pex, that extends the period of
the circadian clock in the cyanobacterium Synechococcus sp. PCC 7942, J. Bacteriol., 180,
2167–2174 (1998).
43. Harmer SL, Panda S, Kay SA, Molecular bases of circadian rhythms, Annu. Rev. Cell Dev. Biol.,
17, 215–53 (2001).
44. Aronson BD, Johnson KA, Loros JJ, Dunlap JC, Negative feedback defining a circadian clock:
autoregulation of the clock gene frequency, Science, 263, 1578–84 (1994).
45. Garceau NY, Liu Y, Loros JJ, Dunlap JC, Alternative initiation of translation and time-specific
phosphorylation yield multiple forms of the essential clock protein frequency, Cell, 89, 469–76
(1997).
46. Konopka RJ, Benzer S, Clock mutants of Drosophila melanogaster, Proc. Natl. Acad. Sci. USA,
68, 2112–16 (1971).
47. Reddy P, Zehring WA, Wheeler DA, Pirrotta V, Hadfield C, Molecular analysis of the period
locus in Drosophila melanogaster and identification of a transcript involved in biological
rhythms, Cell, 38, 701–10 (1984).
48. Gekakis N, Saez L, Delahaye-Brown AM, Myers MP, Sehgal A, Young MW, Weitz CJ, Isolation
of timeless by PER protein interaction: defective interaction between timeless protein and
long-period mutant PERL, Science, 270, 811–15 (1995).
49. Myers MP, Wager-Smith K, Wesley CS, Young MW, Sehgal A, Positional cloning and sequence
analysis of the Drosophila clock gene, timeless, Science, 270, 805–8 (1995).
50. Hardin PE, Hall JC, Rosbash M, Feedback of the Drosophila period gene product on circadian
cycling of its messenger RNA levels, Nature, 343, 536–540 (1990).
51. Sehgal A, Rothenfl¨uh-Hilfiker A, Hunter-Ensor M, Chen YF, Myers MP, Young MW, Rhythmic
expression of timeless: a basis for promoting circadian cycles in period gene autoregulation,
Science, 270, 808–810 (1995).
52. Gonze D, Leloupa JC, Goldbetera A, Theoretical models for circadian rhythms in Neurospora
and Drosophila, Life Sciences, 323, 57-67 (2000).
53. Vatine G, Vallone D, Gothilf Y, Foulkes NS, It’s time to swim! Zebrafish and the circadian clock,
FEBS Let., 585, 1485–1494 (2011).
54. Ben-Moshe Z, Foulkes NS, Gothilf Y, Functional Development of the Circadian Clock in the
Zebrafish Pineal Gland. http://dx.doi.org/10.1155/2014/235781 (2014).
55. Carnevali O, Gioacchini G, Maradonna F, Olivotto I, Migliarini B, Melatonin induces follicle
maturation in Danio rerio, PLoS ONE, 6, Article ID e19978, (2011).
56. Danilova N, Krupnik VE, Sugden D, Zhdanova IV, Melatonin stimulates cell proliferation in
zebrafish embryo and accelerates its development, The FASEB Journal, 18, 751–753 (2004).
57. de Borsetti NH, Dean BJ, Bain EJ, Clanton JA, Taylor RW, Gamse JT, Light and melatonin
schedule neuronal differentiation in the habenular nuclei, Developmental Biol., 358, 251–261
(2011).
58. Piccinetti CC, Migliarini B, Olivotto I, Coletti G, Amici A, Carnevali O, Appetite regulation: the
central role of melatonin in Danio rerio, Hormones and Behav., 58, 780–785 (2010).
59. Rawashdeh O, de Borsetti NH, Roman G, Cahill GM, Melatonin suppresses nighttime memory
formation in zebrafish, Science, 318, 1144–1146 (2007).
174
Oluwatobi Temitope Somade
24
60. Zhdanova IV, Sleep in zebrafish, Zebrafish, 3, 215–226 (2006).
61. Zhdanova IV, Sleep and its regulation in zebrafish, Rev. Neuro., 22, 27–36 (2011).
62. Zhdanova IV, Wang SY, Leclair OU, Danilova NP, Melatonin promotes sleep-like state in
zebrafish, Brain Res., 903, 263–268 (2001).
63. Vatine G, Vallone D, Appelbaum L, Mracek P, Ben-Moshe Z, Lahiri K, et al., Light directs
zebrafish period2 expression via conserved D and E boxes, PLoS Biol., 7, Article IDe1000223
(2009).
64. Ben-Moshe Z, Vatine G, Alon S, Tovin A, Mracek P, Foulkes NS, et al., Multiple PAR and
E4BP4 bZIP transcription factors in zebrafish: diverse spatial and temporal expression
patterns, Chronobiology Int., 27, 1509–1531 (2010).
65. Mracek P, Santoriello C, Idda ML, Pagano C, Ben-Moshe Z, Gothilf Y, et al., Regulation of per
and cry genes reveals a central role for the D-box enhancer in light-dependent gene
expression, PLoS ONE, 7, Article ID e51278 (2012).
66. Gavriouchkina D, Fischer S, Ivacevic T, Stolte J, Benes V, Dekens MPS, Thyrotroph embryonic
factor regulates light-induced transcription of repair genes in zebrafish embryonic cells, PLoS
ONE, 5, Article ID e12542 (2010).
67. Weger BD, Sahinbas M, Otto GW, Mracek P, Armant O, Dolle D, et al., The light responsive
transcriptome of the zebrafish: function and regulation, PLoS ONE, 6, Article ID e17080
(2011).
68. Ripperger JA, Shearman LP, Reppert SM, Schibler U, CLOCK, an essential pacemaker
component, controls expression of the circadian transcription factor DBP, Genes Dev., 14,
679–689 (2000).
69. Ben-Moshe Z, Alon S, Mracek P, Faigenbloom L, Tovin A, Vatine GD, et al., The light-induced
transcriptome of the zebrafish pineal gland reveals complex regulation of the circadian
clockwork by light, Nucleic Acids Res., 42, 3750–3767 (2014).
70. Wager-Smith K, Kay SA, Circadian rhythm genetics: From flies to mice to humans, Nat. Gen.,
26, 23–27 (2000).
71. King DP, Takahashi JS, Molecular genetics of circadian rhythms in mammals, Ann. Rev.
Neuros., 23, 713–742 (2000).
72. Lowrey PL, Takahashi JS, Genetics of the mammalian circadian system: Photic entrainment,
circadian pacemaker mechanisms, and post-translational control, Ann. Rev. Genet., 34, 533–562
(2000).
73. Reppert SM, Weaver DR, Molecular analysis of mammalian circadian rhythms, Ann. Rev.
Physiol., 63, 647–676 (2001).
74. King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, et al., Positional
cloning of the mouse circadian clock gene, Cell, 89, 641–653 (1997).
75. Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, et al., Closing
the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim, Science,
280, 1599–1603 (1998).
76. Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, et al., Role of the Clock
protein in the mammalian circadian mechanism, Science, 280, 1564–1569 (1998).
77. Jin X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ, Reppert SM, A molecular mechanism
regulating rhythmic output from the suprachiasmatic circadian clock, Cell, 96, 57–68 (1999).
78. Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, et al., mCRY1 and mCRY2 are
essential components of the negative limb of the circadian clock feedback loop, Cell, 98, 193–205
(1999).
79. Shearman LP, Jin X, Lee C, Reppert SM, Weaver DR, Targeted disruption of the mPer3 gene:
subtle effects on circadian clock function, Mol. Cell. Biol., 20, 6269–6275 (2000).
80. Bjarnason GA, Jordan R, Circadian variation of cell proliferation and cell cycle protein
expression in man: clinical implications, Prog. Cell Cycle Res., 4, 193–206 (2000).
25
Circadian clock-cell cycle relationships
175
81. Nagoshi E, Saini C, Bauer C, Laroche T, Naef F, Schibler U, Circadian gene expression in
individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter
cells, Cell, 119, 693–705 (2004).
82. Welsh DK, Yoo S, Liu AC, Takahashi JS, Kay SA, Bioluminescence imaging of individual
fibroblasts reveals persistent, independently phased circadian rhythms of clock gene
expression, Curr. Biol., 14, 2289–2295 (2004).
83. Matsuo T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, Okamura H, Control mechanism of the
circadian clock for timing of cell division in vivo, Science, 302, 255–259 (2003).
84. Fu L, Pelicano H, Liu J, Huang P, Lee C, The circadian gene Period 2 plays an important role in
tumor suppression and DNA damage response in vivo, Cell, 111, 41–50 (2002).
85. Gauger MA, Sancar A, Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer,
Cancer Res., 65, 6828–6834 (2005).
86. Oishi K, Miyazaki K, Kadota K, Kikuno R, Nagase T, Atsumi G, et al., Genome-wide expression
analysis of mouse liver reveals Clock regulated circadian output genes, J. Biol. Chem., 278,
41519–41527 (2003).
87. Barnes JW, Tischkau SA, Barnes JA, Mitchell JW, Burgoon PW, Hickok JR, et al., Requirement
of mammalian Timeless for circadian rhythmicity, Science, 302, 439–442 (2003).
88. Unsal-Kacmaz K, Mullen TE, Kaufmann WK, Sancar A, Coupling of human circadian and cell
cycles by the timeless protein, Mol. Cell Biol., 25, 3109–3116 (2005).
89. Gery S, Komatsu N, Baldjyan L, Yu A, Koo D, Koeffler HP, The circadian gene per1 plays an
important role in cell growth and dna damage control in human cancer cells, Mol. Cell., 22,
375–382 (2006).
90. Borgs L, Beukelaers P, Vandenbosch R, Belachew S, Nguyen L, Malgrange B, Cell “circadian”
cycle; New role for mammalian core clock genes, Cell Cycle, 8, 832–837 (2009).
91. Ben-Shlomo R, Kyriacou CP, Circadian rhythm entrainment in flies and mammals, Cell
Biochem. Biophys., 37, 141–156 (2002).
92. Gerard C, Goldbeter A, Entrainment of the mammalian cell cycle by the circadian clock:
modeling two coupled cellular rhythms, PLoS Comput. Biol., 8, Article ID e1002516 (2012).
93. Yang Q, Pando BF, Dong G, Golden SS, van Oudenaarden A, Circadian gating of the cell cycle
revealed in single cyanobacterial cells, Science, 327, 1522–1526 (2010).
94. Ben-Shlomo R. Chronodisruption, cell cycle checkpoints and DNA repair, Ind. J. Exp. Biol., 52,
399–403 (2014).
95. Fu L, Patel MS, Bradley A, Wagner EF, Karsenty G, The molecular clock mediates leptinregulated bone formation, Cell, 122, 803–815 (2005).
96. Hua H, Wang Y, Wan C, Liu Y, Zhu B, Yang C, et al., Circadian gene mPer 2 overexpression
induces cancer cell apoptosis, Cancer Sci., 97, 589–596 (2006).
97. Geyfman M, Kumar V, Liu Q, Ruiz R, Gordon W, Espitia F, et al., Brain and muscle Arnt-like
protein-1 (BMAL1) controls circadian cell proliferation and susceptibility to UVB-induced
DNA damage in the epidermis, Proc. Natl. Acad. Sci. USA, 109, 11758–11763 (2012).
98. Kowalska E, Ripperger JA, Hoegger DC, Bruegger P, Buch T, Birchler T, et al., NONO couples
the circadian clock to the cell cycle, Proc. Natl. Acad. Sci. USA, 110, 1592–1599 (2013).
99. Maier B, Kramer A, A NONO-gate times the cell cycle, Proc. Natl. Acad. Sci. USA, 110, 1565–1566
(2013).
100. Engelen E, Janssens RC, Yagita K, Smits VAJ, van der Horst GTJ, Tamanini F, Mammalian
TIMELESS is involved in period determination and DNA damage-dependent phase advancing
of
the
circadian
clock,
PLoS
ONE,
8,
Article
ID
e56623
(2013)
doi:10.1371/journal.pone.0056623.
101. Levi F, Filipski E, Iurisci I, Li XM, Innominato P, Cross-talks between circadian timing system
and cell division cycle determine cancer biology and therapeutics, Cold Spring Harb. Symp.
Quant. Biol., 72, 465–475 (2007).
176
Oluwatobi Temitope Somade
26
102. Hastings MH, Maywood ES, O’Neill JS, Cellular circadian pacemaking and the role of
cytosolic rhythms, Curr. Biol., 18, R805–R815 (2008).
103. Ben-Shlomo R, Kyriacou CP, Light pulses administered during the circadian dark phase alter
expression of cell cycle associated transcripts in mouse brain, Cancer Genet. Cytogenet., 197,
65–70 (2010).
104. Ben-Shlomo R, Kyriacou CP, Chronodisruption alter expression of tumorigenesis associated
transcripts in mouse brain, WSEAS Transact. Biol. Biomed., 7, 41–50 (2010).
105. Collis SJ, Boulton SJ, Emerging links between the biological clock and the DNA damage
response, Chromosoma, 116, 331–339 (2007).
106. Grechez-Cassiau A, Rayet B, Guillaumond F, Teboul M, Delaunay F, The circadian clock
component bmal1 is a critical regulator of p21waf1/cip1 expression and hepatocyte
proliferation, J. Biol. Chem., 283, 4535–4542 (2008).
107. Yang WS, Stockwell BR, Inhibition of casein kinase 1-epsilon induces cancer-cell-selective,
PERIOD2-dependent growth arrest, Genome Biol., 9, 92 (2008).
108. Dotto GP, p21 (WAF1/Cip1): more than a break to the cell cycle? Biochim. Biophys. Acta.,
1471, 43–56 (2000).
109. Di Cunto F, Topley G, Calautti E, Hsiao J, Ong L, Seth PK, Dotto GP, Inhibitory function of
p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle
control, Science, 280, 1069–1072 (1998).
110. Gartel AL, Tyner AL, Transcriptional regulation of the p21((WAF1/CIP)) gene, Exp. Cell Res.,
246, 280–289 (1999).
111. Pei XH, Xiong Y, Biochemical and cellular mechanisms of mammalian CDK inhibitors: a few
unresolved issues, Oncogene, 24, 2787–2795 (2005).
112. Bjarnason GA, Jordan RC, Sothern RB, Circadian variation in the expression of cell-cycle
proteins in human oral epithelium, Am. J. Pathol., 154, 613–622 (1999).
113. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al., WAF1, a
potential mediator of p53 tumor progression, Cell, 75, 817–825 (1993).
114. Xiang S, Coffelt SB, Mao L, Yuan L, Cheng Q, Hill SM, Period-2: a tumor suppressor gene in
breast cancer, J. Circadian Rhythms, 6, 2–9 (2008).
115. Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S, et al., Role of poly(ADPribose) polymerase (PARP)cleavage in apoptosis. Caspase 3-resistant PARP mutant increases
rates of apoptosis in transfected cells, J. Biol. Chem., 274, 22932–22940 (1999).
116. Castro-Rivera E, Samudio I, Safe S, Estrogen regulation of cyclin D1 gene expression in ZR-75
breast cancer cells involves multiple enhancer elements, J. Biol. Chem., 276, 30853–30861
(2001).
117. Benchimol S, p53-dependent pathways of apoptosis, Cell Death Differ., 8, 1049–1051 (2001).
118. Gery S, Komatsu N, Kawamata N, Miller CW, Desmond J, Virk RK, et al., Epigenetic silencing
of the candidate tumor suppressor gene Per1 in non-small cell lung cancer, Clin. Cancer Res.,
13, 1399–404 (2007).
119. Filipski E, King VM, Li X, Granda TG, Mormont MC, Claustrat B, et al., Disruption of
circadian coordination accelerates malignant growth in mice, Pathol. Biol., 51, 216–219
(2003).
120. Yang X, Wood PA, Oh EY, Du-Quiton J, Ansell CM, Hrushesky WJ, Downregulation of
circadian clock gene Period 2 accelerates breast cancer growth by altering its daily growth
rhythm, Breast Cancer Res. Treat., 117, 423–431 (2009).
121. Lee CC, Tumor suppression by the mammalian Period genes, Cancer Causes Control, 17, 525–530
(2006).
122. Rosbash M, Takahashi JS, Circadian rhythms: the cancer connection, Nature, 420, 373–374
(2002).
27
Circadian clock-cell cycle relationships
177
123. Hua H, Wang Y, Wan C, Liu Y, Zhu B, Wang X, et al., Inhibition of tumorigenesis by
intratumoral delivery of the circadian gene mPer2 in C57BL/6 mice, Cancer Gene Ther., 14,
815–818 (2007).
124. Wood PA, Yang X, Taber A, Oh EY, Ansell C, Ayers SE, et al., Period 2 mutation accelerates
ApcMin/+ tumorigenesis, Mol. Cancer Res., 6, 1786–1793 (2008).
125. Antoch MP, Gorbacheva VY, Vykhovanets O, Toshkov IA, Kondratov RV, Kondratova AA, et
al, Disruption of the circadian clock due to the Clock mutation has discrete effects on aging
and carcinogenesis, Cell Cycle, 7, 1197–1204 (2008).
126. Kondratov RV, Kondratova AA, Gorbacheva VY, Vykhovanets OV, Antoch MP, Early aging
and age-related pathologies in mice deficient in BMAL1, the core component of the circadian
clock, Genes Dev., 20, 1868–1873 (2006).
127. Lowrey PL, Takahashi JS, Mammalian circadian biology: elucidating genome-wide levels of
temporal organization, Ann. Rev. Genomics Hum. Genet., 5, 407–441 (2004).
128. Reddy AB, Wong GK, O’Neill J, Maywood ES, Hastings MH, Circadian clocks: neural and
peripheral pacemakers that impact upon the cell division cycle, Mutat. Res., 574, 76–91
(2005).
129. Lee CC, The circadian clock and tumor suppression by Mammalian period genes, Methods
Enzymol., 393, 852–861 (2005).
130. Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, et al., Coordinated
transcription of key pathways in the mouse by the circadian clock, Cell, 109, 307–320 (2002).
131. Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong WH, et al., Extensive and
divergent circadian gene expression in liver and heart, Nature, 417, 78–83 (2002).
132. Gery S, Gombart AF, Yi WS, Koeffler C, Hofmann WK, Koeffler HP, Transcription profiling of
C/EBP targets identifies Per2 as a gene implicated in myeloid leukemia, Blood, 106, 2827–2836
(2005).
133. Yeh KT, Yang MY, Liu TC, Chen JC, Chan WL, Lin SF, et al., Abnormal expression of period 1
(PER1) in endometrial carcinoma, J. Pathol., 206, 111–120 (2005).
134. Pogue-Geile KL, Lyons-Weiler J, Whitcomb DC, Molecular overlap of fly circadian rhythms
and human pancreatic cancer, Cancer Lett., 243, 55–57 (2006).
135. Chen ST, Choo KB, Hou MF, Yeh KT, Kuo SJ, Chang JG, Deregulated expression of the PER1,
PER2 and PER3 genes in breast cancers, Carcinogenesis, 26, 1241–1246 (2005).
136. Chen-Goodspeed M, Lee CC, Tumor Suppression and Circadian Function, J. Biol. Rhythms,
22, 291–298 (2007).
137. Uchida Y, Hirayama J, Nishina H, A common origin: Signaling similarities in the regulation of
the circadian clock and DNA damage responses, Biol. Pharm. Bull., 33, 535–544 (2010).
138. Kloog I, Haim A, Stevens RG, Barchana M, Portnov BA, Light at night co-distributes with
incident breast but not lung cancer in the female population of Israel, Chronobiol. Int., 25,
65–81 (2008).
139. Kloog I, Stevens RG, Haim A, Portnov BA, Nighttime light level co-distributes with breast
cancer incidence worldwide, Cancer Causes Control, 21, 2059–2068 (2010).
140. Wu J, Dauchy RT, Tirell PC, Wu SS, Lynch DT, Jitawatanarat P, et al., Light at night activates
IGF-IR/PDK1 signaling and accelerates tumor growth in human breast cancer xenografts,
Cancer Res., 71, 2622–2631 (2011).
141. Kantermann T, Roenneberg T, Is light-at-night a health risk factor or a health risk predictor?
Chronobiol. Int., 26, 1069-1074 (2009).
142. Schernhammer ES, Kroenke CH, Laden F, Hankinson SE, Night work and risk of breast cancer,
Epidemiology, 17, 108–111 (2006).
143. Straif K, Baan R, Grosse Y, Secretan B, El Ghissassi F, Bouvard V, et al., Carcinogenicity of
shift-work, painting, and fire-fighting, Lancet Oncol., 8, 1065–1066 (2007).
178
Oluwatobi Temitope Somade
28
144. Parent ME, El-Zein M, Rousseau MC, Pintos J, Siemiatycki J, Night work and the risk of cancer
among men, Am. J. Epidemiol., 176, 751–759 (2012).
145. Menegaux F, Truong T, Anger A, Cordina-Duverger E, Lamkarkach F, Arveux P, et al., Night
work and breast cancer: A population-based case-control study in France (the CECILE
study), Int. J. Cancer, 132, 924–931 (2013).
146. Kamdar BB, Tergas AI, Mateen FJ, Bhayani NH, Oh J, Night-shift work and risk of breast
cancer: a systematic review and meta-analysis, Breast Cancer Res. Tr., 138, 291–301 (2013).
147. Greene MW, Circadian rhythms and tumor growth, Cancer Lett., 318, 115–123 (2012).
148. Levi F, Okyar A, Dulong S, Innominato PF, Clairambault J, Circadian timing in cancer
treatments, Annu. Rev. Pharmacol. Toxicol., 50, 377–421 (2010).
149. Levi F, Altinok A, Clairambault J, Goldbeter A, Implications of circadian clocks for the
rhythmic delivery of cancer therapeutics, Phil. Trans. R. Soc. A., 366, 3575–3598 (2008).
150. Wulff K, Gatti S, Wettstein JG, Foster RG, Sleep and circadian rhythm disruption in psychiatric
and neurodegenerative disease, Nat. Rev. Neuro., 11, 589–599 (2010).
151. Feuvray D, Circadian rhythms and cardiovascular disease, Heart Metab., 44, 3–4 (2009).
152. Martino TA, Oudit GY, Herzenberg AM, Tata N, Koletar MM, Kabir GM, et al., Circadian
rhythm disorganization produces profound cardiovascular and renal disease in hamsters,
Amer. J. Physiol. Reg. Int. Comp. Physiol., 294, 1675–1683 (2008).