REVIEWS
THE CIRCADIAN CLOCK:
PACEMAKER AND TUMOUR
SUPPRESSOR
Loning Fu and Cheng Chi Lee
The circadian rhythms are daily oscillations in various biological processes that are regulated by
an endogenous clock. Disruption of these rhythms has been associated with cancer in humans.
One of the cellular processes that is regulated by circadian rhythm is cell proliferation, which often
shows asynchrony between normal and malignant tissues. This asynchrony highlights the
importance of the circadian clock in tumour suppression in vivo and is one of the theoretical
foundations for cancer chronotherapy. Investigation of the mechanisms by which the circadian
clock controls cell proliferation and other cellular functions might lead to new therapeutic targets.
Department of Molecular
and Human Genetics,
Baylor College of Medicine,
One Baylor Plaza, Houston,
Texas 77030, USA.
Correspondence to C.C.L.
e-mail: [email protected]
doi:10.1038/nrc1072
350
The word ‘circadian’ is derived from a Latin phrase
meaning ‘about a day’. Daily oscillation of physiological
and behavioural processes in plants and animals have
been reported since the time of Alexander Great, in the
fourth century BC. However, it wasn’t until the middle of
the last century that such oscillating rhythms were found
to be driven by an internal timing machine — the ‘circadian clock’ — which can maintain biological rhythms of
about 24 hours in the absence of external cues1.
Mutagenic studies in fruitfly were the first to indicate
that the circadian clock could be regulated genetically2.
The first circadian gene, Period, was cloned from fruitflies
in the mid-1980s (REFS 3,4). Since then, the rapid advances
in the field of circadian biology research have revealed
that these clocks are operated by numerous gene products that function in interacting feedback loops in all
species studied. Circadian genes have been discovered in
all species studied, although they might have developed
following independent evolutionary pathways in different
kingdoms5. Clocks provide organisms with a survival
advantage, by organizing their behaviour and physiology
around cyclic changes in the environment.
Circadian rhythms regulate hundreds of functions in
the human body, including sleep and wakefulness, body
temperature, blood pressure, hormone production,
digestive secretion and immune activity. Disruption of
these rhythms can have a profound influence on our
health. For example, disruption of circadian rhythms has
been linked to insomnia, jet lag, stomach ailments, coronary heart attacks and depression6, and is commonly
observed among cancer patients7,8.
There has been a long history of research seeking a
link between circadian clocks and tumour suppression. Studies of animal models and human tumour
samples have revealed that the disruption of circadian
rhythms is an important endogenous factor that contributes to mammalian cancer development9–12. CANCER
CHRONOTHERAPY is based on the asynchrony that exists
in cell proliferation and drug metabolism between
normal and malignant tissues. Administration of cancer therapy based on circadian timing has shown
encouraging results, but still lacks a strong mechanistic foundation13. Further investigations into the molecular link between the circadian clock and growth
regulation will generate novel therapeutic strategies
for improving the efficacy of cancer treatment. What
is our current understanding of the role of the circadian clock in growth control, and how does it affect
tumour suppression and cancer treatment?
Circadian clockwork
Eight core circadian genes have been identified so
far. They are Clock14, casein kinase Iε (CKIε)15,16,
cryptochrome 1 (Cry1) and chryptochrome 2 (Cry2)17–19,
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Summary
• The circadian clock is the internal timing machine that can sustain rhythms of about
24 hours in the absence of external cues. The circadian clock is operated by the
feedback loops of the circadian genes in the mammalian central pacemaker, as well as
in most peripheral tissues.
• The mammalian central pacemaker is located in the suprachiasmatic nuclei (SCN) of
the brain and controls the activity of peripheral clocks through the neuroendocrine
and autonomic nervous systems. The circadian clock regulates hundreds of functions
in the human body.
• Disruption of circadian rhythms has been linked to mammalian tumorigenesis and
tumour progression, and has been used as an independent prognostic factor of
survival time for patients with certain metastatic cancers.
• Normal and malignant tissues often show asynchronies in cell proliferation and
metabolic rhythms. Based on these observations, cancer chronotherapy has been
developed to improve the efficacy in cancer treatment and the quality of patients’ life.
• The circadian clock functions in vivo as a tumour suppressor at the systemic, cellular
and molecular levels. The central clock is capable of generating 24-hour cellproliferation rhythms in peripheral tissues through the activity of the neuroendocrine
and autonomic nervous systems.
• Molecular clocks in peripheral tissues control cell-proliferation rhythms by regulating
the expression of cell-cycle genes. The core circadian genes are also involved in
regulating cell proliferation. The circadian clock in peripheral tissues responds directly
to DNA damage and could be important in the control of the cell cycle and apoptosis.
• The molecular clockworks and cell-cycle clocks in peripheral tissues can be regulated
simultaneously by the central clock, through interacting signalling pathways.
Further study of the mechanism of the circadian clock in tumour suppression and
the DNA-damage response has important implications for cancer therapy.
Period1 (Per1), Period2 (Per2) and Period3 (Per3)20–24,
and Bmal1 (REFS 25,26). The three Per genes encode
PER–ARNT–SIM (PAS)-domain proteins that function
in the nucleus but apparently do not directly bind to
DNA. The PAS domains are functionally important, as
they provide surfaces that allow heterodimerization
among different clock proteins. The Clock and Bmal1
genes encode basic–helix–loop–helix (bHLH)-PAS
transcription factors. The levels of mRNAs and proteins
of these circadian genes, except those of Clock and CK1ε,
oscillate throughout the 24-hour period27 (TABLE 1).
The molecular clockwork of the mammalian central
pacemaker has been the subject of several recent
reviews5,27–29. Briefly, it can be explained by a model of
transcription-translation feedback loops of circadian
genes (FIG. 1). The anatomy of the mammalian circadian
clock contains three components: input pathways, the
central pacemaker and output pathways. The input
pathways transmit information from environmental
cues to the central pacemaker. The central pacemaker
synchronizes with the environment to generate endogenous rhythms. The output pathways convert the instructions from the central pacemaker into daily oscillations
in various physiological and behavioural processes27,28
(FIG. 2). The central pacemaker in mammals resides in the
SUPRACHIASMATIC NUCLEI (SCN) of the anterior hypothalamus30,31 (BOX 1). The SCN is composed of multiple, single-cell circadian oscillators that, when synchronized,
generate coordinated circadian outputs32,33. Ablation of
SCN leads to loss of circadian rhythm in rodents30,31.
Among all environmental cues, light is the most
powerful circadian synchronizer34,35. In mammals, the
circadian photoreception pathways are distinct from
those of visual perception. Mice lacking rods and cones
respond normally to light-induced melatonin
suppression and PHASE SHIFTS in behaviour rhythms 36,37.
Table 1 | Mammalian circadian regulators
Circadian regulators Role in mammalian circadian clock
Direct and indirect targets
in growth regulation
References
Period 1 (Per1)
Involved in circadian phase resetting in SCN
and in peripheral tissues; mutations shorten
circadian period in rodents
Period 2 (Per2)
Mutation alters behaviour rhythmicity,
results in neoplastic growth phase and
deficient DNA-damage response in rodents,
and causes advanced sleep disorder in
humans; stimulates Bmal1 expression
Period 3 (Per3)
Mutations do not affect behaviour rhythmicity
in rodents
Casein kinase Iε (CKIε)
Phosphorylates Per to control Per stability
and nuclear localization; mutations alter
behaviour rhythmicity in rodents
β-Catenin
Clock
Physically associates with Bmal1; binds to
E-box sequences to stimulate Per and Cry
transcription; mutations alter behaviour
rhythmicity in rodents
c-Myc?
Bmal1
Physically associates with Clock; binds to
E-box sequences to stimulate Per and Cry
transcription; mutations alter behaviour
rhythmicity in rodents
c-Myc
11,25,26,30,31
123,140
Cry1 and Cry2
Physically associate with and stabilize
Per; mutations alter behaviour rhythmicity
in rodents; might also be important in
photoreception; suppress Bmal1/Clock
transcription activity
c-Myc
11,17–19,31
NATURE REVIEWS | C ANCER
20–22,57,123,
131–133,136,141
c-Myc, cyclin D1, p53,
Mdm2, cyclin A, Gadd45α
11,22,24,60,197
23
15,16,173–175
14,31,140
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Rev-Erbα
Bmal1
Rev-Erbα
Bmal1
Cry
Cry
Bmal1
Bmal1
Rev-Erbα
Clock
Per
Clock
Per
Per Cry
Per CKIε
Per Cry
CKIε
Nucleus
CKIε
Per
P
Degradation
Cytoplasm
Figure 1 | Mammalian core circadian gene feedback loops. Circadian rhythms are
generated by the feedback loops of the core circadian genes. In the SCN neurons, the
intracellular levels of Clock remain steady throughout the 24-hour period, whereas Bmal1
expression levels are high at the beginning of a subjective day and low at the beginning of a
subjective night. The high level of Bmal1 promotes the formation of Bmal1–Clock
heterodimers. These bind to E-box sequences in the promoters of the Cry, Per and Rev–Erbα
genes to activate transcription at the beginning of a circadian day. The Bmal1–Clock
heterodimer can also inhibit Bmal1 transcription. After transcription and translation, the
Rev–Erbα protein enters the nucleus to suppress the transcription of Bmal1 and Cry genes.
As the Per proteins, such as Per2, accumulate in the cytoplasm, they become
phosphorylated (P) by CKIε. The phosphorylated forms of Per are unstable and are degraded
by ubiquitylation. Late in the subjective day, however, Cry accumulates in the cytoplasm,
promoting the formation of stable CKIε/Per/Cry complexes, which enter the nucleus at the
beginning of a subjective night. Once in the nucleus, Cry1 disrupts the Clock/Bmal1associated transcriptional complex, resulting in the inhibition of Cry, Per and Rev–Erbα
transcription, and derepression of Bmal1 transcription193,194. It is not clear whether Per and
Cry must dissociate from the CKIε/Per/Cry complex to inhibit the activity of Clock/Bmal1
heterodimer and to stimulate Bmal1 transcription in the nucleus. The interacting positive and
negative feedback loops of circadian genes ensure low levels of Per and Cry, and a high level
of Bmal1 at the beginning of a new circadian day5,27–29. Solid lines indicate direct regulation,
and dashed lines indicate indirect regulation.
SUPRACHIASMATIC NUCLEI
(SCN). The mammalian master
circadian clock. The SCN are
small bilateral structures located
next to the third ventricle and
just above the optic chiasm in
mammalian brain. Each SCN
nucleus contains about 10,000
neurons that are synchronized to
generate coordinated circadian
outputs in vivo.
PHASE SHIFT
The displacement of waveform
in time. When a waveform is
displaced by a complete
wavelength, it is described as
having a phase shift of 360
degrees. When a waveform is
displaced by a half a wavelength,
it is described as having a phase
shift of 180 degrees.
352
However, the phases of circadian gene expression in
peripheral tissues are delayed by a few hours, relative
to those of SCN, and ablation of SCN abolishes circadian gene oscillation in peripheral tissues49,50. So, the
peripheral clocks are either driven or synchronized by
the SCN pacemaker. Under certain conditions, however, such as when food supply is restricted at resting
phase, the peripheral clocks can be completely
uncoupled from the SCN central clock, and take their
phasing cues from the feeding time rather than from
the SCN51,52.
Certain human blind subjects with no significant
perception of light still retain the circadian response
to light38. Light signals are received by a subset of
MELANOPSIN-EXPRESSING RETINAL GANGLION CELLS, and are
transmitted directly to the SCN through the retinohypothalamic tract (RHT)39–42 (BOX 1). In addition, the
mammalian circadian genes Cry1 and Cry2 are also
involved in photoreception by certain retinal neurons43.
The RHT produces neurotransmitters that activate a
cascade of events in SCN neurons, leading to circadian
phase resetting27,28.
Peripheral clocks. Circadian rhythms are regulated in
peripheral tissues by similar interacting loops of core
circadian gene products. These ‘peripheral clocks’ are
regulated by the SCN pacemaker, through both the
autonomic nervous system (ANS) and neuroendocrine
systems44–46 (BOX 1). The rhythmic expression of core
circadian genes is observed in most peripheral tissues47,48, and can be induced in cultured fibroblasts49.
Clock control. Recently, it was shown that the transcription feedback loops of circadian genes can be
regulated by intracellular redox pathways53. This indicates that peripheral clock timing can be affected by
the intracellular metabolic rate, which is independent
of the SCN pacemaker 54. The phase resetting in
peripheral tissues by restricted feeding is a relatively
slow process that can be quickly reversed when food
is provided with a normal feeding schedule. The
phase reversal of the peripheral clock seems to be
under the control of hormones of the neuroendocrine system. Hormones such as glucocorticoids
inhibit the uncoupling of peripheral clocks from the
central pacemaker55.
The SCN central clock and peripheral tissue clocks
both regulate cell functions by controlling the expression of clock-controlled genes. These genes are not,
however, required for clock function. Whereas some
clock-controlled genes are regulated indirectly by the
molecular clock, some are regulated directly, such as
by the Bmal1–Clock heterodimer, which binds to
E-box sequences in gene promoters11,27. Recent studies
indicate that 2–10% of all mammalian genes are
clock-controlled genes55–61. Most of these show tissue/organ-specific expression patterns and are
involved in organ function. Only a small set of clockcontrolled genes are expressed in multiple organs.
Among them are genes that encode key regulators of
cell-cycle progression11,56–59.
Loss of circadian rhythm and tumorigenesis
The finding that disruption of circadian rhythms led to
increased mammary tumour development was first
reported in the 1960s (REFS 62,63). These studies indicated
that disruption of circadian endocrine rhythms — either
through constant light exposure or by PINEALECTOMY —
accelerates breast epithelial stem-cell proliferation,
induces mammary-gland development and increases the
formation of spontaneous mammary tumours in
rodents64,65. In addition, light-induced circadian-clock
suppression also increases carcinogenesis in rodents66,67.
Several epidemiological studies have revealed a role
for the circadian clock in human breast cancer development9,10,68,69. They showed that disruption of circadian cycles, such as in people that work predominantly
at night, is a risk factor for breast cancer development.
The breast cancer risk increased with the number of
years, or number of hours per week, that individuals
spent working at night. These studies were carefully
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Awake
Sleep
Aldosterone
Cortisol
Urine output
Awake
Awake
Sleep
Lymphocytes
Monocytes
Platelets
Eosinophils
Figure 2 | Circadian regulation of hormones, urine and the
immune system. The circadian oscillation of hundreds of
biological processes enables a human body to adapt to 24hour light/dark cycles. a | The serum levels of cortisol and
aldosterone, and urine volume195, oscillate in circadian cycles in
humans. The cortisol level peaks in healthy individuals at early
morning and reach their lowest levels before bedtime.
Disruption of cortisol circadian rhythm can result in fatigue and
restlessness, weight loss, insomnia and coronary heart
disease. Aldosterone is a steroid hormone that is secreted by
the cortex of the adrenal gland and regulates the body’s
electrolyte balance. The level of aldosterone is normally low
during the day and high during sleep in humans. Disruption of
aldosterone rhythms result in sodium and water retention,
increased blood pressure and coronary heart disease. Urine
volumes have inverted circadian oscillating patterns, compared
to that of aldosterone, in healthy humans. b | The circadian
oscillation of lymphocyte, monocyte, platelet and eosinophil
levels in healthy young adults. The activity of the immune
system, usually represented by the number of lymphocytes,
peaks in the late evening and is lowest in the early morning.
Disruption in this circadian rhythm could lead to immune
suppression. Adapted from REF. 113.
MELANOPSIN-EXPRESSING
RETINAL GANGLION CELLS
A small subset of retinal
ganglion cells that are
intrinsically photosensitive and
express the opsin-like protein
melanopsin. These neurons
project directly to the
suprachiasmatic nucleus of the
mammalian central circadian
clock, as well as to the
intergeniculate leaflet and the
olivary pretectal nucleus in the
brain. Mice that are deficient in
melanopsin show attenuated
responses to light stimuli.
PINEALECTOMY
Ablation of the pineal gland.
The pineal gland is a cone-shape
gland that is located at the
posterior end of the third
ventricle in the brain. The pineal
gland produces melatonin, a
hormone that is important for
regulating circadian rhythmicity
in humans. The level of
melatonin rises at night and falls
during the day.
designed, and included a large number of participants,
as well as long follow-up periods. The study by
Schernhammer et al.9 included 78,562 women from
the Nurses’ Health Study and involved a 10-year follow-up period. So, circadian rhythms could be more
important than family history in determining breast
cancer risk70.
Disruption of circadian rhythm not only increases
the risk of tumour development, but also accelerates
cancer progression in tumour-bearing animals and in
cancer patients. Carcinoma- or sarcoma-bearing rats
showed increased tumour growth and reduced survival time when kept in alternative light/dark (L/D)
cycles — such as L/D for 14:10 hours followed by D/L
for 10:14 hours every 3 days — than those kept in
constant L/D cycles of 12:12 hours71. A recent study
showed that ablation of SCN in mice resulted in loss
of circadian rhythm in wheel-running activity, body
temperature, plasma corticosterone and lymphocyte
numbers. Osteosarcoma or pancreatic adenocarcinoma both grew 2–3 times faster in mice bearing
SCN lesions than in controls, leading to significant
reductions in survival time12.
Similar observations have been made in cancer
patients. When circadian rhythms — determined by
levels of salivary cortisol — were measured in patients
with metastatic breast cancer, early mortality was
observed more frequently in patients who had lost normal diurnal salivary cortisol variation. After adjusting
for other factors that might affect survival time, the circadian rhythm of salivary cortisol remained as a statistically significant predictor of survival time for patients
with breast cancer72. The circadian rhythm of rest/activity was also monitored in patients with metastatic colorectal cancer. The 2-year study showed that patients
with clearly defined rest and activity rhythms had a fivefold higher survival rate and experienced significantly
less fatigue than patients who had lost rest/activity
rhythm. So, the rest/activity rhythms provide a novel
independent prognostic factor for survival and tumour
response of patients with metastatic colorectal cancer73.
Chronotherapy
Most conventional cancer therapeutic strategies are
aimed primarily at maximizing cytotoxicity and avoiding acquired drug resistance, whereas the ability of the
host biological system to cooperate with the therapy
has not been taken into full consideration. Anticancer
drugs usually act selectively on proliferating cells or at a
specific phase of the cell cycle. These drugs not only target cancer cells, but also normal host tissues that are
engaged in active cell proliferation. So, the balance
between the level of damage to normal tissues and the
targeting efficiency to tumours, or the ‘therapeutic
index’, is not always favourable. Chronotherapy has
been developed in an attempt to improve the efficacy of
cancer treatment and the quality of patients’ life.
The principle of chronotherapy in oncology is to
take advantage of the asynchronies in cell proliferation and drug metabolic rhythms between normal
and malignant tissues by administering therapy at a
specific time of the day. This could potentially minimize the damage to host tissues and maximize drug
toxicity to tumours13,74. The efficiency of anticancer
drugs in vivo is determined by their absorption, distribution, intracellular metabolism and elimination. All
these processes show circadian variation in vivo 75.
Some of them, such as the rhythm of tumour blood
flow, can be distinct from that of normal tissue 76.
Once inside the cell, the effect of a cytotoxic drug is
mainly determined by the circadian phase of that cell’s
proliferation cycle74,77,78.
Circadian variation of mitotic activity in normal
human tissues was first described in 1938 (REF. 79). It is
now well known that cell proliferation in rapidly renewing mammalian tissues follows daily oscillation patterns
in vivo77,80,81. The mitotic rhythms of mammalian cancers
have been studied since 1940 (REF. 82). So far, accumulated
evidence indicates that mammalian tumours, even at
advanced stages, are not temporally disorganized masses.
The proliferation of tumour cells can be stably entrained
to the host circadian rhythm or follow a tumour-specific
rhythm, in vivo. The coupling of proliferating rhythm
between host and tumour cells is usually observed in
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Box 1 | The mammalian central clock and how it controls peripheral tissues
Circadian rhythms are generated by a small subset of neurons that are located in the suprachiasmatic nuclei (SCN),
which is located in the anterior hypothalamus in the brain30,31. The longitudinal view of mouse brain (a) illustrates the
direct light input pathway from the eye to the SCN, and the direct targets of SCN fibres. Light signals are received by a
small group of melanopsin-expressing retinal ganglion cells in the eye and transmitted to the SCN neurons directly
through the retinothypothalamic tract (RHT)39–42. The retinal ganglion cells also project to the intergeniculate leaflet
(IGL) and the oliveray pretectal nucleus in the brain, which transmit light signals indirectly to the SCN40 (not shown).
The SCN central pacemaker controls physiological and behavioural rhythms through diffusible molecules that are
produced by the SCN neurons 188–190, as well as by targeting other regions of the brain directly. Four different targets of
SCN fibres have been identified in rodent brain. Three of them are confined within the medial hypothalamus. They are
the endocrine neurons that produce corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH)
and gonadotropin-releasing hormone (GnRH); the autonomic paraventricular neurons (aPVN); and the intermediate
neurons. Intermediate neurons include the neurons in the subparaventricular nucleus of the hypothalamus (sPVN), the
neurons of the dorsomedial nucleus of the hypothalamus (DMH), and the neurons of the medial preoptic nucleus
(MPN, not shown). Outside of the hypothalamus, the SCN neurons project to the paraventricular nucleus of the
thalamus (PVT) and the IGL191. A cross-section of mouse brain (b) shows the location of the SCN nuclei. The SCN are
small bilateral structures located aside the third ventricle (3V) and just above the optic chiasm. Each SCN nucleus
contains about 10,000 neurons that are synchronized to generate coordinated circadian outputs in vivo 32,33. The SCN
controls the circadian rhythmicity of plasma corticosterone production through the neuroendocrine and autonomic
nervous systems in mice (c). The SCN controls the activity of the autonomic nervous system through the autonomic
neurons of the paraventricular nucleus (aPVN). These neurons project from the hypothalamus to preganglionic
parasympathatic and sympathetic neurons in brainstem nuclei, such as the dorsal motor nucleus of the vagus (DMV)
and in the intermediolateral cell columns (IML) of the spinal cord44. The SCN indirectly controls the secretion of
adrenocorticotropic hormone (ACTH) from the pituitary gland, through the CRH-producing endocrine neurons in the
hypothalamus192. Circadian variations in the activity of the autonomic nervous system and in ACTH production result
in rhythmic release of corticosterone from the adrenal gland into the blood. So, plasma levels of corticosterone undergo
a 24-hour circadian variation in vivo. See REFS 44 and 99 for indepth reviews on the neuroanatomy output pathways of
SCN to peripheral tissues. LV, lateral ventricle. Part a is modified from REF. 99.
a
b
Forebrain
LV
Cerebellum
Hypothalamus
3v
PVT
sPVN
aPVN
DMH
Optic
chiasm
IGL
GnRH
SCN
TRH
Light
CRH
SCN
Optic nerve
Pituitary gland
RHT
Eye
c
CRH
aPVN
DMV
SCN
Spinal
cord
IML
Pituitary gland
Light
ACTH
Adrenal
gland
Corticosterone
354
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Stress, social environment
Light
Other regions of the brain
SCN
Pineal gland
Hypothalamus
Autonomic
nervous
system
Pituitary gland
Melatonin
Glucocorticoid
Interferons
Gonads
Adrenal gland
Interleukin-1
Peripheral tissues
Cell proliferation
and apoptosis
Figure 3 | The circadian clock controls cell proliferation
and apoptosis at the systemic level. Light and other
environmental cues reach the suprachiasmatic nuclei (SCN)
through various input pathways. The SCN clock
synchronizes with the environment to generate endogenous
rhythms, which are transmitted through output pathways to
peripheral tissues. Representative output pathways, such
as the autonomic nervous system (ANS), the
hypothalamic–pituitary–gonadal (HPG) and the
hypothalamic–pituitary–adrenal (HPA) axes, are shown. The
pineal gland and peripheral tissues can also feed back to
SCN or HPA axes, through production of melatonin, to
regulate homeostasis. Melatonin binds to receptors on SCN
neurons to induce phase shifts196. The adrenal glands
produce glucocorticoids, which have negative feedback on
the hypothalamus to terminate the release of corticotropinreleasing hormone117. The products of immune activity, such
as interferon-α and -γ, and interleukin-1, can also modulate
the activity of SCN, as well as the HPA axis116,118. Feedback
pathways are indicated by dashed lines.
slow-growing tumours, although, in these cases, DNA
synthesis and mitotic indices are often considerably
higher in tumour cells throughout the 24-hour
period83–85. The altered circadian rhythms, or ultradian
rhythms (less than 24-hour oscillation), in cell proliferation are often observed in fast-growing or
advanced-stage tumours85–89. So, cancer treatment can
be optimized by exploring the cytokinetic asynchrony
between tumour and host tissues, and applying anticancer drugs at a time of the day that is associated with
maximal tumour susceptibility and host tolerability74.
The efficacy of chronotherapy with various anticancer drugs was first tested in animal models. These
studies indicated that both in vivo host tolerability and
drug efficacy were affected by circadian rhythms, and
the best therapeutic index was achieved by coupling
treatment with these rhythms90–94. The results of the animal model studies have been extrapolated to randomized clinic trials of patients undergoing treatment for
advanced-stage cancers74,95–97. Some of these clinical trials involve more than 2,000 patients, mostly with
metastatic colorectal cancer97. The results of these studies indicate that chronotherapy reduced drug toxicity
and improved patients’ performance. Chronotherapy
was two- to eightfold more effective than conventional
therapy — patients showed improvement in tumour
response rate and the duration of the response, and
decreased frequency of tumour metastasis74, although it
did not increase the long-term survival of patients with
metastatic colorectal or breast cancer. Chronotherapy
has, however, been shown to significantly increase the
survival time of children with acute lymphoblastic
leukaemia (ALL) — this approach increased survival
time by 4.2-fold in an 8-year study, and by 2.6-fold in a
15-year disease-free survival analysis98.
Circadian clocks as tumour suppressors
So what is the mechanism by which the circadian
clock affects tumour growth? The circadian clock has
been shown to function as a tumour suppressor at the
systemic, cellular and molecular levels in vivo.
At the systemic level. The SCN central clock regulates
cell proliferation and apoptosis in peripheral tissues
through the ANS and neuroendocrine systems, such as
the hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes44–46,99 (FIG. 3).
In vivo, the ANS innervates all peripheral tissues, except
skeletal muscle. It also controls cell proliferation and
death in innervated tissues and organs in a tissue- or
cell-type-specific manner through G-protein-coupled
transmembrane-receptor-mediated pathways100–106.
Hormones produced by the HPA and HPG axes, such
as oestrogen and glucocorticoids, are widely known to
control cell proliferation and apoptosis in peripheral
tissues107–111. The activity of the ANS and neuroendocrine systems is regulated by the SCN clock, and
shows a 24-hour rhythmic activity in vivo45, providing
an explanation for the circadian-coordinated cell-proliferation rhythm in peripheral tissues. Disruption of
ANS and neuroendocrine rhythms could lead to
deregulation of cell-proliferation rhythm in peripheral
tissues and promote oncogenesis112.
The ANS and neuroendocrine system also function at the systemic level to suppress tumour development through immunomodulation (FIG. 3). Both
ANS and hormones of the HPA axis, such as glucocorticoids, have been shown to control cytokine production, leukocyte distribution, proliferation and
apoptosis. This means that the immune response is
also regulated by the central circadian clock 113.
Disruption of circadian rhythms could therefore lead
to immune suppression, which could disrupt cancer
immunosurveillance and promote tumour development 12,71,102–104,111–115. Immune products such as
cytokines can also act to modulate the activity of the
SCN clock and the HPA and HPG axes, providing an
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immune-regulatory, circadian-paced feedback loop116–118.
So, by anticipating and adapting to external and internal
cues, the SCN clock controls overt rhythmicity in cell
proliferation in peripheral tissues in vivo.
At the cellular and molecular level. In cells of peripheral tissues, the SCN’s clock controls cell proliferation
and apoptosis by regulating the expression of circadian-controlled genes. Recent studies have shown that
about 7% of all clock-controlled genes that have been
identified in rodents regulate either cell proliferation
or apoptosis56–59. These clock-controlled genes include
c-Myc and Mdm2, the tumour-suppressor genes Trp53
and Gadd45α, as well as genes that encode the caspases, cyclins, transcription factors, and ubiquitinassociated factors that are involved in regulating the
cell cycle and apoptosis11,56–59. The rhythmic expression
of several cyclins, as well as that of the tumour suppressor p53, is also observed in human oral mucosa.
These expression patterns are synchronized with the
circadian oscillation patterns of Per1 and Bmal1
expression in the same tissue119,120.
Apart from controlling the expression of cell-cycle
genes and tumour-suppressor genes at the transcriptional and post-transcriptional levels, the core circadian genes are also involved directly in modulating
the intracellular signalling pathways that regulate cell
proliferation. Recently, it has been shown that the
core circadian regulator CKIε also functions in promoting cell proliferation by stabilizing β-catenin.
Overexpression of CKIε mimics the effect of WNT
signalling, resulting in cytoplasmic accumulation of
β-catenin and its subsequent nuclear localization 121–123. β-Catenin interacts with transcription
factors of the T-cell-specific transcription factor/lymphoid enhancer factor-1 (TCF/LEF) family to regulate transcription124 and promote tumorigenesis125.
Genes activated by β-catenin/TCF/LEF include members of the AP1 transcription family, c-Jun and Fra1
(REFS 126,127), and c-Myc and Ccnd1 (which encodes
cyclin D1) (REFS 128–130). It is not known whether the
role of CKIε in cell proliferation is independent of its
role in circadian-clock function.
Interestingly, β-catenin is destabilized by glycogen
synthase kinase-3β (Gsk3β) in the absence of WNT
signalling124. Gsk3β is a functional homologue of the
core circadian gene Shaggy in fruitfly131. Although
direct molecular evidence for the role of Gsk3β in
mammalian circadian-clock function has not been
shown, by antagonizing CKIε’s ability to promote
β-catenin stability, Gsk3β could also be involved in circadian control of cell-cycle progression. Therefore, as
the molecular clockworks regulate the cell cycle and
apoptosis in cells of peripheral tissues, mutations in circadian genes could conceivably result in deregulation of
these processes and tumour development.
Per2 regulation of p53 and c-Myc. Mice with disruptions
in the core circadian gene Per2 have recently been
shown to display salivary-gland hyperplasia and develop
spontaneous lymphoma11. It is likely that deregulation
356
of multiple molecular pathways contribute to the cancer-prone phenotype of the Per2-mutant mice.
Deregulation of the Myc-mediated growth-regulatory
pathway is one possible mechanism by which disruption of the circadian clock could promote tumour formation. The expression pattern of c-Myc mRNA shows
a low-amplitude circadian oscillation in all mouse tissues studied, but expression is significantly increased
throughout the 24-hour period in Per2 mutants. In
addition, the c-Myc P1 promoter is suppressed directly
by the core circadian regulators, indicating that c-Myc is
a clock-controlled gene. The expression of Myc-target
genes Ccns1 and Ccna1/2 also show circadian oscillation
patterns in vivo, and this oscillation is significantly
altered following Per2 mutation11.
Per2-mutant mice are more susceptible to lymphoma after exposure to γ-radiation. This observation
might be explained by the fact that loss of Per2 activates
the Myc signalling pathways (FIG. 4) that induce cell proliferation and apoptosis. Induction of cell-cycle entry by
c-Myc also sensitizes cells to apoptosis. Suppression of
c-Myc-induced apoptosis, such as by co-expression of
Bcl-xL, is sufficient to promote tumour progression
without additional oncogenic mutations132. One of the
key mediators in c-Myc-induced apoptosis is the
tumour suppressor p53 (REF. 133). Loss of p53 activity is
necessary and sufficient for c-Myc-accelerated lymphomagenesis in mice134,135. The Per2-mutant thymocytes are also deficient in p53-mediated apoptosis in
response to γ-radiation. So, deregulation of c-Myc and
deficiency in p53-mediated apoptosis are likely to
underlie the high incidence of radiation-induced lymphoma in Per2-mutant mice11 (FIG. 4). As the core circadian genes show coordinated expression in vivo, the
model in which a c-Myc signalling pathway mediates
circadian control of cell proliferation needs to be tested
in additional animal models, such as in mice that are
deficient in other core circadian genes.
There has been a long debate about whether the
circadian clock and the cell-cycle clock are connected
in vivo. In our experiments, γ-radiation-induced apoptosis is circadian time-dependent in both wild-type and
Per2-mutant thymocytes. When irradiated at the early
stage of active phase or at the early stage of resting
phase, Per2-mutant thymocytes show a G2/M-specific
resistance to radiation-induced apoptosis11. So, the circadian clock not only regulates the expression of cellcycle genes — it could also be involved in controlling
cell-cycle checkpoint function.
In DNA-damage response. The core circadian genes
respond directly to γ-radiation. Disruption of Per2
abolishes the response of all core circadian genes to
γ-radiation. So, the molecular clock itself can be modulated by genotoxic stress in peripheral tissues. The ability of circadian genes to mediate the DNA-damage
response seems to be cell autonomic, as Per2-mutant
thymocytes have attenuated p53 induction in response
to γ-radiation in vitro11. It has been shown recently that
the clock genes also respond to low levels of ultraviolet
(UV) irradiation in cultured human keratinocytes136.
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REVIEWS
Per2
γ-irradiation
Bmal1/Clock
p53
Apoptosis
or
Bmal1/Npas2
c-Myc
Genomic
instability
Genomic instability,
cell proliferation
Cancer
Figure 4 | A model for the role of Per2 in tumour
suppression. Core circadian gene products regulate a
number of oncogenes, such as c-Myc. Overexpression of
c-Myc has been shown to lead to DNA damage, hyperplasia
and tumorigenesis. Heterodimeric circadian regulators such
as Bmal1–Clock and Bmal1–Npas2 negatively regulate c-Myc
expression at the transcriptional level. Loss of Per2 function
reduces Bmal1 expression throughout 24-hour light/dark
cycles, leading to decreased intracellular levels of
Bmal1–Npas2 or Bmal1–Clock, and derepression of c-Myc.
Following γ-irradiation, the loss of Per2 function partially
impairs p53-mediated apoptosis, leading to genomic
instability and accumulation of damaged cells. These cells can
still progress through the cell cycle in the presence of genomic
DNA damage, due to the high level of c-Myc expression,
resulting in tumour formation after γ-radiation. Solid lines
indicate the pathways that have been demonstrated by recent
studies. The dashed line indicates a regulatory pathway(s) that
is still not fully understood11.
The molecular pathways by which the circadian
clock controls the DNA-damage response in peripheral
tissues remain unclear. It has recently been shown
that casein kinase II (CKII) is involved in controlling
circadian rhythms in Arabidopsis, Neurospora and
Drosophila137–140. In fruitflies, CKIIα directly phosphorylates PER, which regulates its ability to enter the
nucleus140 (FIG. 1). In mammals, CKII regulates cell-cycle
progression by phosphorylating and activating c-Myc.
The interaction of CKII and c-Myc has been associated
with various mammalian cancers141–143. CKII also phosphorylates p53 in response to UV irradiation144. It is
important to determine whether CKII functions in the
mammalian circadian clock, and whether it is involved
in the circadian-clock-controlled DNA-damage
response in vivo.
Peripheral clock control
A brief light pulse at the beginning of a subjective
night activates a cascade of events in the SCN neurons
, such as activation of expression of the immediateearly genes c-Fos and JunB, as well as the circadian
genes Per1 and Per2 (REFS 145–147). Light exposure at the
end of a subjective night, however, only induces Per1
(REF. 47). So, the response of the SCN central clock to
light stimuli is circadian time-dependent. Light stimuli
at the beginning of a subjective night results in the
release of intracellular Ca2+ in SCN neurons, which, in
turn, activates signalling pathways, such as the mitogen-activated protein kinase (MAPK)/extracellularsignal-regulated kinase (ERK) pathway, and the calcium/calmodulin and c-AMP-protein kinase A (PKA)
pathways (FIG. 5). Light exposure at the end of a subjective night activates nitric oxide (NO) and c-GMP
pathways148–150. The activation of these pathways leads
to phosphorylation of c-AMP/calcium-responsive element-binding protein and subsequent activation of
Per1 via a c-AMP-responsive element in the promoter
of the Per1 gene. Activation of Per1 is involved in the
light-induced phase resetting in the SCN clock47,148–152.
Mammalian peripheral clocks do not respond directly
to light stimuli, but are instead regulated by cyclic changes
in the levels of neurotransmitters, growth factors, and
hormones such as glucocorticoids and retinoids55,153,154.
These control expression of the core circadian genes Per1,
Per2 and Cry1 throughout the 24-hour period153.
Retinoids interact with nuclear receptors, such as retinoid
X receptor-α (RXRα) and retinoic-acid receptor-α
(RARα), to regulate the transcriptional activity of
Clock/Bmal1 (REF. 154). Glucocorticoids interact with glucocorticoid receptors (GRs), and localize to the nucleus to
regulate the expression of genes that contain glucocorticoid-responsive elements (GREs)155. The Per1 gene contains two almost-perfect GREs — one in its 5′ promoter
and another in its first intron156.
A phase shift in the expression patterns of the core circadian genes Per1, Per2 and Cry1 can be induced by
restricting nutrient supply55,153. Oscillations in circadian
gene expression can also be induced in cultured rat
fibroblasts and NIH3T3 cells by serum shock, or by
treatment with dexamethasone or tetradecanoylphorbol
13-acetate (TPA). This induction does not require
de novo protein synthesis, but is mediated directly
through various signalling pathways, such as the c-AMP,
protein kinase C (PKC), Ca2+ and MAPK pathways157,158
(FIG. 5). The Ras–MAPK signalling pathway has been
shown to control circadian output pathways in fly and
chick159,160. It is likely that similar pathways are involved in
maintaining peripheral clock control in mammals.
The c-AMP–PKA, PKC and MAPK pathways are
well known for their roles in regulating cell proliferation. The MAPK family contains three well-characterized subfamilies of kinases. They are the ERKs, the
c-JUN amino-terminal kinases (JNKs) and the p38
MAPKs161. Among these three subfamilies, the ERK signalling pathway controls cell proliferation and apoptosis
in response to diverse stimuli, such as growth factors,
cytokines and carcinogens. It also responds to polypeptide hormones and neurotransmitters through interactions with the c-AMP signalling pathway106,161–164 (FIG. 5).
Deregulation of ERK pathways is commonly
observed in human cancer cells, and inhibitors of
these pathways have been tested as anticancer agents
in clinical trials165–168. ERK signalling leads to the activation of AP1 transcription factors, which are dimeric
basic leucine zipper proteins from the Jun and Fos
families169. The AP1 transcription factors regulate a
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REVIEWS
Growth
factors
Neurotransmitters,
peptide hormones
Glucocorticoids
Ras
GRs
cAMP
PKA
Akt
Rap1
Raf1
Gsk3β
JNK
Braf
MEK
β-Catenin
CKIε
MAPK1, 2
CREB
ERK
TCF/LEF
Core circadian
genes
+
AP-1
–
c-Myc, cyclin D1,
p53
Cell-cycle progression
or apoptosis
Figure 5 | Signalling pathways linking the circadian
clock to cell-cycle regulation in peripheral tissues.
Production of extracellular signals is regulated by the central
clock, and can result in cell-cycle progression or apoptosis
in peripheral tissues. For example, circadian-regulated
production of growth factors activates the mitogenactivated protein kinase (MEK, MAPK1,2)/extracellularsignal-regulated kinase (ERK) pathways, whereas
neurotransmitters and peptide hormones regulate the
c-AMP/protein kinase A (PKA) signalling pathways106,161–164.
These pathways interact through Rap1, which inhibits Ras
signalling, and Braf, which activates Raf1 signalling in a celltype-dependent manner. Signalling through the c-AMP/PKA
and MEK pathways, however, leads to activation of the
c-AMP response-element binding protein (CREB). CREB
activates the transcription of core circadian genes in
peripheral tissues157,158. Additionally, ERK signalling
activates the transcription factor AP-1, which regulates
production of c-Myc, cyclin D1 and p53, and thereby
mediates cell proliferation and apoptosis169. The c-AMP
pathway also indirectly promotes β-catenin stability, through
AKT and glycogen synthase kinase-3β (Gsk3β) signalling.
The core circadian gene product CKIε also stabilizes
β-catenin, and thereby activates TCF/LEF (T-cell-specific
transcription factor/lymphoid enhancer factor-1)121–123,162.
This leads to activation of c-Myc and cyclin D1.
Glucocorticoids can inhibit AP-1 activity directly through
ligand-bound glucocorticoid receptors (GRs), or indirectly
by inhibiting the c-JUN amino-terminal kinase (JNK)
signalling pathway180–184. So, peripheral clocks and cellcycle clocks are intimately linked to each other by
interacting signal-transduction pathways.
358
wide range of cellular processes, including cell proliferation, apoptosis and differentiation. Genes that are
directly or indirectly controlled by AP1 include
Ccnd1, Trp53, Cdkn1a and Cdkn2a (REFS 170–173).
Persistent activation of AP1 leads to abnormal
cell-cycle progression and transformation169.
It is unlikely that AP1 controls circadian gene expression in peripheral tissues, however, as the induction of
c-Fos and Per1 occur at about the same time in SCN,
and mice deficient in c-Fos show normal Per1 induction
by light146,174. It is most likely that induction of the circadian genes Per1 and Per2, along with the immediate
early-response genes c-Fos and c-Jun, is regulated simultaneously through interacting signalling pathways in
peripheral tissues.
In addition to controlling circadian rhythm in
peripheral tissues, retinoids and glucocorticoids also
regulate cell proliferation. These factors are therefore
used as cancer therapeutics. The retinoid-acid receptors RARα, RARβ and RXR induce G1 cell-cycle
arrest by suppressing Ccnd1 expression175–177, and disruptions in RARα are associated with acute promyelocytic leukaemia178. GR signalling activates genes that
inhibit components of the ERK pathway179. The GR
also interacts directly with AP-1, which blocks
the ability of AP-1 to activate transcription 180.
Glucocorticoids can also modulate c-AMP and
MAPK signalling through non-genomic mechanisms181–184. Glucocorticoids inhibit cell proliferation
in many types of cells by promoting apoptosis and
cell-cycle arrest, which are often correlated with the
downregulation of c-Myc and cyclin D3 (REFS 185–187).
Together, this evidence indicates that molecular
clockworks and cell-cycle clocks in peripheral tissue are
regulated by a complex interaction of pathways that
include glucocorticoids, retinoids, c-AMP, PKC, WNT,
Ca2+ and MAPK signalling153,154,157,158,161–164,175–177,179,181
(FIG. 5). In vivo, production of extracellular signals —
such as growth factors, cytokines, neurotrasmitters and
hormones — are controlled by the SCN central
clock45,99,102,104,113 (FIG. 3). The SCN therefore controls the
24-hour rhythmic activities in peripheral tissues, by
controlling intracellular signalling (FIG. 5). The peripheral
clocks, synchronized by the central clock and operating in their own local environments, respond to these
signals to regulate the genes that control cell-cycle progression, such as c-Myc, Ccnd1 and Trp53 (REFS
11,119,120). However, under certain conditions, such as
in the case of DNA damage, the peripheral clocks
respond immediately to the damage, and could then
operate independently of the central pacemaker, to
control local cell-cycle checkpoints, activate apoptosis
and suppress malignant growth11.
Future directions
A large amount of in vivo evidence has shown that
the circadian clock is involved in tumour suppression, and that cancer should no longer be treated as a
local disorder. However, the ability of the biological
clock to suppress malignant growth and to cooperate
with cancer treatment has not been fully explored.
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© 2003 Nature Publishing Group
REVIEWS
Future research should be focused on studying the
mechanisms by which the circadian clock controls cell
proliferation, apoptosis and responses to genotoxic
stress. Advanced cancers are often autonomic in cellproliferation rhythm. It will be important, therefore, to
study how tumour-cell proliferation escapes the control of the central pacemaker, and whether somatic
mutations in clock genes or deregulation of entraining
pathways for peripheral clocks are involved. This information is particularly needed to improve the efficacy
of current chronotherapy. In addition, as slow-growing tumours can fall under the control of the host
circadian clock, it will be important to study whether
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Online links
DATABASES
The following terms in this article are linked online to:
Cancer.gov: http://www.cancer.gov/cancer_information/
acute lymphoblastic leukaemia | breast cancer | colorectal cancer |
osteosarcoma | pancreatic cancer
LocusLink: http://www.ncbi.nih.gov/LocusLink/
β-catenin | Bcl-xL | Bmal1 | Ccna1 | Ccna2 | Ccnd1 | CKÎε | Cry1 |
Cry2 | cyclin D3 | Fos | Fra1 | Gadd45α | Gsk3β | Jun | JunB |
MAPK | Mdm2 | Myc | Per1 | Per2 | Per3 | PKC | Ras | Trp53
FURTHER INFORMATION
A time to heal – chronotherapy tunes in to body’s rhythms:
Http://www.fda.gov/fdac/features/1997/397_chrono.html
Biological clocks: http://www.seoulin.co.kr/Up/clock/clock.html
Biotiming tutorial:
http://www.cbt.virginia.edu/tutorial/HISTBACK.html
From circadian rhythm to cancer therapy:
http://www.eortc.be/home/chrono/Fromcircadianrhythms.html
Access to this interactive links box is free online.
VOLUME 3 | MAY 2003 | 3 6 1
© 2003 Nature Publishing Group