311

In Vitro Cell. Dev. Biol.—Animal 41:311–320, November–December 2005

q

2005 Society for In Vitro Biology

1071-2690/05

$18.00+0.00

CLOCK GENES OF MAMMALIAN CELLS: PRACTICAL IMPLICATIONS

IN TISSUE CULTURE

BERTRAND KAEFFER

1

AND

LISSIA PARDINI

CRNH de Nantes, Institut National Recherche Agronomique, Unite´ Fonctions Digestives et Nutrition Humaine, BP 71627 44316,

NANTES, Cedex 03, France

(Received 30 June 2005; accepted 14 September 2005)

S

UMMARY

The clock genes family is expressed by all the somatic cells driving central and peripheral circadian rhythms through

transcription/translation feedback loops. The circadian clock provides a local time for a cell and a way to integrate the
normal environmental changes to smoothly adapt the cellular machinery to new conditions. The central circadian rhythm
is retained in primary cultures by neurons of the suprachiasmatic nuclei. The peripheral circadian rhythms of the other
somatic cells are progressively dampened down up to loss unless neuronal signals of the central clock are provided for
reentrainment. Under typical culture conditions (obscurity, 37

6

1

8

C, 5–7% CO

2

), freshly explanted peripheral cells

harbor chaotic expression of clock genes for 12–14 h and loose, coordinated oscillating patterns of clock components.
Cells of normal or cancerous phenotypes established in culture harbor low levels of clock genes idling up to the reoc-
currence of new synchronizer signals. Synchronizers are physicochemical cues (like thermic oscillations, short-term ex-
posure to high concentrations of serum or single medium exchange) able to reinduce molecular oscillations of clock
genes. The environmental synchronizers are integrated by response elements located in the promoter region of period
genes that drive the central oscillator complex (CLOCK:BMAL1 and NPAS2:BMAL1 heterodimers). Only a few cell lines
from different species and lineages have been tested for the existence or the functioning of a circadian clockwork. The
best characterized cell lines are the immortalized SCN2.2 neurons of rat suprachiasmatic nuclei for the central clock
and the Rat-1 fibroblasts or the NIH/3T3 cells for peripheral clocks. Isolation methods of fragile cell phenotypes may
benefit from research on the biological clocks to design improved tissue culture media and new bioassays to diagnose
pernicious consequences for health of circadian rhythm alterations.

Key words: clock; circadian rhythm; primary culture; established cell line

I

NTRODUCTION

Circadian rhythms are 24-h periods or cycles of physiology and

behavior, generated by endogenous genetic feedback loops occur-
ring in a majority of eukaryotes and some prokaryotes (Dunlap,
1999; Harmer et al., 2001). All mammalian cells investigated to
date seem to possess internal circadian clocks that subsist when
cells from the central nervous system or peripheral tissues are ex-
planted in primary cultures or subcultured as established or im-
mortalized cell lines (Tsuchiya and Nishida, 2003) suggesting that
the basic clock mechanism is intrinsic and self-sustained. In phys-
iology, the circadian clock, also called circadian oscillator, is an
internal device that keeps the body’s time by driving and/or coor-
dinating a circadian rhythm. The concept has been integrated in
the definition of homeostasis, which is the capacity of a living struc-
ture not only to withstand and adapt its internal conditions to pro-
gressive or sudden changes of the environment but also to anticipate
the occurrence of reoccurring events (i.e., reactive homeostasis;
Moore-Ede, 1986). The human circadian clock and its functioning
in central or peripheral tissues are currently explored to increase

1

To whom correspondence should be addressed at E-mail: kaeffer@

nantes.inra.fr

the therapeutic efficacy of timed administration of drugs or radiation
and to offer better advice on lighting and meal timing useful for
frequent travelers suffering from jet lag and for night workers’ com-
fort. At the molecular level, oscillators are molecules that are in-
teracting to create a pacemaker. The pacemaker is an internal
mechanism that keeps time for the circadian clock by either coor-
dinating or constructing rhythms (i.e., patterns of reoccurring events
that can be loosely thought of as cycles). Besides circadian rhythms,
there are other rhythms in cell biology that are called ultradian,
which refer to cycles that are shorter than 20 h (i.e., the beating of
heart or of cardiomyocytes in culture), and infradian, which are
cycles that are greater than 28 h (i.e., intestinal crypts renewal in
5–6 d in humans). The mammalian circadian system consists of at
least two major oscillator systems, one entrainable by light, called
central clock, and located in the suprachiasmatic nuclei (SCN) and
another by food that is anatomically and functionally distinct but
of unknown location. The mammalian genome encodes at least a
dozen of genes directly involved in the regulation of the feedback
loops constituting the circadian clock. The positive limb of the loop
is driven by two transcription factors, CLOCK (King et al., 1997;
Gekakis et al. 1998; or a close analog called NPAS2 [Reick et al.,
2001]) and BMAL1 (Hogenesch et al., 1998; Gekadis et al., 1998),

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KAEFFER AND PARDINI

which are basic helix–loop–helix proteins with a PER-ARNT-SIM
(PAS) domain.

A first negative limb involves the protein regulators PERIOD 1

(Sun et al., 1997; Tei et al., 1997), PERIOD 2 (Albrecht et al.,
1997; Shearman et al., 1997), or PERIOD 3 (Zylka et al., 1998).
These proteins do not directly bind to DNA, but they have a PAS
domain to build complexes with CRYPTOCHROME 1 or 2 (Griffin
et al., 1999; Kume et al., 1999; van der Horst et al., 1999). The
proteins CASEIN KINASE I epsilon or delta are involved in the
turnover of PERIOD molecules by phosphorylating these proteins
and regulating their nuclear translocation (Shearman et al., 2000;
Vielhaber et al., 2000; Camacho et al., 2001; Akashi et al., 2002;
Eide et al., 2002; Lee et al., 2004, Takano et al., 2004). A second
negative limb involves members of the retinoic acid–related orphan
nuclear receptors, RORA (Sato et al., 2004b) and REV-ERB-AL-
PHA (Preitner et al., 2002), which repress and modulate bmal1
expression. An additional loop of regulation driven by two differ-
entiated embryon chrondrocyte proteins (Honma et al., 2002; Ham-
aguchi et al., 2004; Sato et al., 2004a) has been proposed. These
oscillator systems control the activity of clock-controlled genes that
function in the rate-limiting steps of various biological pathways
(Kornmann et al., 2001; Panda et al., 2002; Storch et al., 2002;
Ueda et al., 2002; Duffield, 2003). In the absence of any environ-
mental cue, the protein PERIOD2 is believed to reboost the cir-
cadian cycle by stimulating the transcription of bmal1 gene leading
to the dimerization with CLOCK and to the reinduction of a new
circadian cycle (Oishi et al., 1998a, 1998b, 2003; Zheng et al.,
1999, 2001; Shearman et al., 2000; Bae et al., 2001; Lee et al.,
2001; Yu et al., 2002). The circadian regulation of period2 gene
has also been linked in vivo to tumor suppression and DNA damage
response (Fu et al., 2002). Period1 and 2 genes are molecular can-
didates to study the adaptive response of the cellular metabolism
with environmental stressors, and they have been associated with
the signaling pathway allowing a cell to anticipate and integrate
changes in its normal environment. More studies on the circadian
rhythmicity of clock genes will help improve our understanding of
the adaptation of cells to in vitro conditions of culture as well as
design new bioassays to unravel the pernicious consequences on
animal and human health of circadian rhythm alterations by envi-
ronmental cues.

The aim of this review is to underline the benefits of research on

the biological clock’s in practical cell culture.

C

IRCADIAN

R

HYTHMICITY IN

P

RIMARY

, E

STABLISHED

,

AND

I

MMORTALIZED

C

ELLS

Most mammalian cells are believed to harbor functional clock

genes, but effective oscillations are under the control of environ-
mental cues called synchronizers. Different levels of in vitro dem-
onstration have been reached, ranging from the existence of the
molecular clock components in a particular cell line to the extensive
explorations of the conditions to reinduce clockwork into cultured
cells. We are introducing the main results obtained on cultures of
suprachiasmatic neurons and on the few cell lines from different
species and lineages that have been tested for the existence or the
functioning of a peripheral circadian clockwork.

Persistence of a circadian rhythmicity in primary cultures of neu-

rons from the central pacemaker (suprachiasmatic nuclei). In mam-
mals, the hypothalamic suprachiasmatic nucleus is the center of

circadian rhythms, such as sleep–wakefulness, glucose utilization,
and hormonal rhythms. Neurons in the SCN have the remarkable
ability to autonomously generate a cycle of electrical activity with
a period very close to 24 h and to retain clock gene products os-
cillating in vitro under an environmental light cue. Suprachiasmatic
cells dispersed in a culture dish and maintained in primary cultures
will continue to oscillate individually, showing a cyclic pattern of
clock gene expression with a period of approximately 24 h. With
primary cultures of period-1-luciferase–transfected neurons isolated
from the SCN, Yamazaki et al. (2000) have recorded light emission
during weeks in culture without dampening. A similar demonstra-
tion has been seen with period1-luciferase–transfected neurons iso-
lated from the SCN of mouse (Asai et al., 2001).

In vivo, immediate early gene expression in the SCN is tightly

correlated with entrainment of SCN-regulated rhythms (Ginty et al.,
1993), and the alternation of dark and light is perceived by melan-
opsin cells of the retina and transferred by neuronal connections
toward the SCN neurons with glutamate or serotonin as mediators
(Berson et al., 2002). In culture, glutamate triggers an intracellular
Ca

11

increase on primary cultured suprachiasmatic neurons (Hon-

ma et al., 1998), circadian firing, and clock gene oscillations in
immortalized neurons from the suprachiasmatic nuclei (Hurst et al.,
2002b).

Neurons of the suprachiasmatic nuclei are considered to be the

site of the central clock regarding synchronization by lightning. But
gonadotropin-secreting cell lines (Gillespie et al., 2003) have been
found to retain the melatonin circadian rhythmicity. As a recent
demonstration has shown that drosophila’s clock is driven by two
subpopulations of neurons retaining oscillations in antiphase (Grima
et al., 2004), we cannot entirely exclude the possibility of other
mammalian cells able to retain and drive circadian rhythmicity
linked to either the alternation of dark and light cycles, the core
temperature regulation, or the physiology and behavior of feeding.

Pseudoperiodic induction of molecular oscillations of clock genes

in peripheral cells. Under typical culture conditions (obscurity, 37

6

1

8

C, 5–7% CO

2

), most cells may retain a very low expression

of clock genes (Fig. 1A). Experiments have been performed with
peripheral cells using a serum shock as synchronizer to trigger
pseudoperiodic oscillation waves with a maximum of three consec-
utive cycles and a phase shift of the oscillation by repeated appli-
cation of stimuli (Balsalobre et al., 1998). Similar experiments were
conducted with many primary cultures from laboratory rodents: mu-
rine cerebellar granule neurons (Akiyama et al., 2001) and embry-
onic fibroblasts sampled at day 13 (Yagita et al., 2001; Pando et
al., 2002), rat smooth muscle cells of the vasculature isolated from
the thoracic aorta (Nonaka et al., 2001), and human samples: me-
lanocytes, keratinocytes, dermal and neonatal foreskin (Zanello et
al., 2000), and vasculature smooth muscle cells (McNamara et al.,
2001; Curtis et al., 2004).

In established or immortalized cell lines, the reinduction of clock

components as well as their progressive dampening over time are
dependent on the cell line itself, its lineage, as well as its organ of
origin. The immortalized peripheral cells include few cell lines de-
rived from laboratory rodents: Rat-1 fibroblasts (Balsalobre et al.,
1998, 2000a, 2000b; Yagita and Okamura, 2000; Yagita et al.,
2000, 2001; Brown et al., 2002; Duffield et al., 2002; Hirota et al.,
2002), Rat 3Y1 embryonic fibroblasts (Grundschober et al., 2001),
INS1 rat insulinoma beta-cells (Mu¨hlbauer et al., 2004), NIH3T3
cells (Akashi et al., 2000, 2002; Allen et al., 2001; Oh-hashi et

313

CLOCK GENES OF MAMMALIAN CELLS

al., 2002; Jung et al., 2003; Tsuchiya et al., 2003; Kaasik and Lee,
2004), and Swiss 3T3 and N2A neuroblastoma cells (Chilov et al.,
2001). In humans, experiments have been reported with the SH-
SY5Y (Maronde and Motzkus, 2003), the fetal lung WI38 (Miyazaki
et al., 2003, 2004), 293 kidney cells (Chilov et al., 2001), and Kelly
neuroblastoma and colon carcinoma Caco-2 and HT-29 clone 19A
(Pardini et al., 2004, 2005).

In summary, the best-characterized cell lines are the immortal-

ized SCN2.2 neurons of rat suprachiasmatic nuclei for the central
pacemaker and the rat-1 fibroblasts or the NIH/3T3 cells for pe-
ripheral clocks. Sex (Abizaid et al., 2004) and aging (Yamazaki et
al., 2002) are known to affect circadian rhythmicity, and the ac-
quisition of clock genes by the embryo has been described on pri-
mary cultures of mouse embryo fibroblasts from embryonic day 13
(Yagita et al., 2001; Pando et al., 2002). But functional clock genes
expressed by the embryonic stem cells of hematopoeitic origin are
not mentioned (Li and Akashi, 2003), and there is no cellular model
to study the acquisition of a functional clock between the two-cell
and the 16-mm stage of embryonic development.

Entrainment of circadian rhythm in peripheral cells cocultured

with suprachiasmatic neurons. Suprachiasmatic cells of fetal rat pro-
genitors immortalized by E1A adenovirus oncogene retaining cir-
cadian rhythmicity have been established as the SCN2.2 cell line
(Earnest et al., 1999a) (Fig. 1B). These cells retain both molecular
and metabolic oscillations (Earnest et al., 1999b; Allen et al., 2001)
as well as components of multiple circadian regulatory pathways
(Hurst et al., 2002a). Under serum stimulation, NIH/3T3 fibroblasts
are displaying molecular oscillations of clock genes but not of met-
abolic oscillations of compounds like 2-deoxy-glucose putatively
driven by clock-controlled genes (Allen et al., 2001). The possibility
to coculture in double-chamber immortalized suprachiasmatic cells
with epithelial cells has opened the way for the in vitro manipula-
tion of the SCN-communicated signal that is necessary for the gen-
eration of metabolic rhythmicity in NIH/3T3 cells (Allen et al.,
2001), but the cross-species transmission of entraining signals from
these immortal SCN cells has not yet been demonstrated.

In conclusion, there may be tissue-specific differences in the

molecular composition of the circadian clock, and clock components
that have subtle effects on the central clock function may play a
more prominent role in the regulation of peripheral clocks. Yagita
et al. (2001) have used spontaneously immortalized mouse embryo
fibroblasts to explore the main clock components (proteins and
mRNA), suggesting that peripheral clocks in cultured cells may be
similar in composition and regulation as central clocks. This hy-
pothesis is not exactly true, as circadian clocks differ in some clock
components, in their coupling with the different categories of syn-
chronizers, as well as in their output. The most striking example is
the apparent redundancy of clock with its homolog npas2, which
are largely equivalent molecules with slight biological differences
(Reick et al., 2001; Rutter et al., 2001). In parallel experiments,
Pando et al. (2002) have shown that period1-deficient mouse em-
bryo fibroblasts placed in culture display an intrinsic period of only
20 h instead of 23 h in the tissue of an intact animal, which can
be rescued by reimplantation in a mouse, implying that the function
of PERIOD1 is different in SCN neurons and in peripheral tissues.
The next sections will illustrate the clock functioning in cultured
cells and the influence of synchronizers and their practical conse-
quences in tissue culture.

C

IRCADIAN

C

LOCK

F

UNCTIONING IN

T

ISSUE

C

ULTURE

After their explantation in primary culture, peripheral cells

should adapt to new culture conditions. Rats transgenic for period1
gene coupled to a luciferase reporter gene have been used as a
source of tissue explants. Light emission by liver explants has been
recorded over 4 d (Stokkan et al., 2001) and found highly variable
the first 12 to 14 h after explantation. A periodic oscillation of light
emission by these explants is recorded from the first subjective day
(12–36 h after explantation) to the third day. In similar experiments,
Yamazaki et al. (2000) have reported that lung, muscle, and liver
explants can be reentrained after dampening of their circadian
rhythmicity by SCN signals. These works suggest that the molecular
clockwork is progressively dampened in cultured cells that harbor
low levels of clock genes idling up to the reoccurrence of new syn-
chronizer signals. For most cell lines, manipulating the circadian
clock means interfering with the regulation of period genes and
appreciating the capacity of the molecular clockwork to be coupled
or uncoupled with the cellular physiology.

Period genes may integrate circadian environmental cues to syn-

chronize intracellular networks to a cyclic environment. The environ-
mental synchronizers are integrated by response elements located
in the promoter region of period genes that drive the central oscil-
lator complex. The period genes are members of the immediate early
gene family because cells like human normal fibroblasts exposed to
cycloheximide, an inhibitor of transcription, retain a response to-
ward stressful conditions characterized by a dramatic increase in
PERIOD proteins (Miyazaki et al., 2004).

Functional mapping of period gene promoters were conducted

with cell lines transfected with period1 linked to a luciferase re-
porter gene (rat1 fibroblasts, ovine pars tuberalis, HUH-7 hepatoma
cells). Period1 gene is known to contain five well-conserved E-boxes
(CACGTG) elements on which CLOCK: BMAL1 or NPAS2: BMAL1
dimers can bind (Hida et al., 2000) along with three C/EBP-ele-
ments that are possible mediators of the IL-6 effect (Motzkus et al.,
2002), three CAAT-, and one AP-1-like element (Motzkus et al.,
2002). Several studies have been conducted on period1 regulation
by PKA and PKC activation (Akashi and Nishida, 2000; Motzkus
et al., 2000; Yagita and Okamura, 2000) and by interleukin-6
through the CCAAT/enhancer binding protein-beta (C/EBP-beta) re-
sponse elements present in the hPER1 promoter sequence (Motzkus
et al., 2002). The promoters of period1 and 2 genes (but not of
period3) contain a cAMP-responsive element (called CRE) that
binds to CREB proteins. These CRE sites are integrating the cAMP
response to a wide category of synchronizers (like serotonin, glu-
tamate, calcium ions, and light) as well as the response to a second
wide category of synchronizers (like growth factors, hormones, and
cytokines) acting through the extracellular signal regulated kinase
leading to the mitogen-activated kinase pathways (independently of
the CLOCK: BMAL1 activity (Travnickova-Bendova et al., 2002).

The establishment of an alternating regime of period gene acti-

vation by CLOCK:BMAL1 heterodimers and PERIOD-dependent
inhibition of this activation, responsible in turn for the rising and
falling phases of the circadian oscillations in the levels of period
transcripts, is considered central to the mechanism of circadian
oscillations (Gekakis et al., 1998; Shearman et al., 2000). The ex-
pression of murine period in the suprachiasmatic nuclei has been
found more important as the entry point into the loop for resetting
stimuli with murine cryptochrome 1 defining the phases of rhythmic

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KAEFFER AND PARDINI

F

IG

. 1.

Conditions to reinduce molecular oscillations of clock components in tissue culture. Somatic cells remain entrainable by

external cues in culture depending on their tissular origin and on the composition of the medium. (A) Synchronization in typical tissue
culture conditions to reinduce pseudoperiodic molecular oscillations of clock components like period2 mRNA shown here in colon
carcinoma cell monolayers (Caco-2; mean of triplicate) with a period of 26 h for the cell population to compare with the reported period
of 25.65 h for culture of primary fibroblasts isolated from mPER2::LUC-SV40 knock-in mice with individual cell period range from 22.4
to 29.7 h (Welsh et al., 2004). (B) Coculture between fibroblasts and neurons from the suprachiasmatic nuclei to reentrain circadian
oscillations of clock components as reported by Allen et al. (2001). (C) Improvement of isolation procedures of fragile phenotypes from
the intestinal epithelium like the human colonic crypt by manipulating period 1 and 2, members of the immediate early-gene family.

315

CLOCK GENES OF MAMMALIAN CELLS

←

Several physicochemical parameters (temperature, nutrients, oxygen, or carbon dioxide tensions) have been explored but not an oscillating
pattern close to the in vivo conditions like the thermic oscillations described by Brown et al. (2002). Figure is published in color online
at http://inva.allenpress.com/invaonline?/request

5index.html.

outputs dependent on the suprachiasmatic nuclei (Reddy et al.,
2002; Hastings et al., 2003). In cultured cells, most experiments
are conducted without exposition to any neuronal signals; as a con-
sequence, coupling between the circadian clockwork and cellular
physiology have to be interpreted carefully as pseudoperiodic os-
cillations of clock products for the cell population. Work on primary
fibroblasts and Rat-1 (Nagoshi et al., 2004; Welsh et al., 2004) as
well as NIH3T3 fibroblasts (Nagoshi et al., 2004) has shown that
in vitro dampening of clock’s products may be the consequence of
a progressive desynchronization of many independent fibroblastic
oscillators. However, these models cannot explain the capacity of
populations of suprachiasmatic neurons to remain synchronized in
vitro. More work should be done on the paracrine signaling between
cells using the system proposed by Allen et al. (2001) (Fig. 1B)
with another cell lineage like epithelial cells, which retain the ca-
pacity to produce in culture polarized cell monolayers with func-
tional gap junctions.

Coupling and uncoupling with cell proliferation, differentiation,

and senescence. The expression of clock genes in association with
specific cell cycle phases has been detected on biopsies of human
oral mucosa (Bjarnason et al., 2001; Bjarnason and Jordan, 2002).
The CLOCK:BMAL1 complex acts on E-box (CACGTG) elements
of target genes. Three E-boxes elements were found within the 1.2
kb of the mouse wee1 gene 5

9

-upstream region (Matsuo et al.,

2003). As seen with the drug retrorsine, a plant alkaloid that is a
strong inhibitor of cyclins, the circadian oscillatory mechanism in
proliferating hepatocytes does not require the help of a cell cycle
for its normal functioning (Matsuo et al., 2003). The mechanism of
this uncoupling with cellular proliferation remains unknown.

The aging and the senescent cells are the only ones in which a

loss of circadian clock control has been reported. In aged WI-38
cells, Miyazaki et al. (2004) have reported that PERIOD proteins
do not undergo circadian oscillation in cells that have exceeded 40
population doublings. In senescent colonocytes at the top of the
human crypt, Pardini et al. (2005) have shown a loss of PERIOD1
polarization.

Acquisition of a cancerous phenotype may lead to some alteration

in circadian rhythmicity due to mutation or loss of some molecular
components or to more subtle alterations in input and ouput genes
of clocks. The pseudoperiodic oscillations of clock proteins and
their importance in day-to-day cultures of immortal cell lines may
be considered negligible, but this is an unexplored field with the
potential for discoveries related to the regulation of circadian
rhythms in cancer cells.

Coupling and uncoupling of the circadian clocks with cellular

metabolic networks. In vitro analyses indicate that the core com-
ponents of these molecular feedback loops regulate downstream
clock-controlled genes, such as arginine vasopressin, thyrothropic
embryo factors, EBP4, and albumin D binding protein via interac-
tion of CLOCK (or NPAS2) and BMAL1 dimers with E-box regu-
latory sequences (Gekakis et al., 1998; Jin et al., 1999; Ripperger
et al., 2000; Mun˜oz et al., 2002) as well as two neuropeptides that

are candidate output factors: transforming growth factor-alpha (Kra-
mer et al., 2001) and prokineticin-2 (Cheng et al., 2002).

The whole transcriptome is believed to be under a circadian mas-

ter clock in the prokaryote Synechoccocus elongatus (Liu et al.,
1995), but the question is still open for eukaryotes. Only 85 cir-
cadian regulated transcripts have been detected on cultured Rat
3Y1 embryonic fibroblasts (Grundschober et al., 2001) and 41 tran-
scripts on rat-1 fibroblasts (Duffield et al., 2002). In rat 3Y1 em-
bryonic fibroblasts, only dbp displayed a cyclic expression pattern
for more than one cycle. This may indicate that these genes have
a circadian expression profile in vivo that is not maintained in cul-
tured cells. According to these divergent data, only 10% of the
eukaryote transcriptome is believed to be clock dependent (Akhtar
et al., 2002; Hastings et al., 2003), leaving few if any metabolic
pathways out of the control of clockwork.

In tissue culture, a direct application of lactate to the medium

of cultured neuroblastoma cells activates NPAS2:BMAL1-depen-
dent gene expression (Rutter et al., 2001), suggesting that the cir-
cadian clock may be linked to the cellular response to NADP(H)
redox state (Rutter et al., 2002).

The demonstration of metabolic oscillations on many cell lines

is generally not attempted in part because of the difficulty defining
and, as such, detecting the circadian rhythmicity of mRNA or pro-
tein.

N

ATURE AND

S

TRENGTH OF

S

YNCHRONIZERS

In conventional cell culture, the inputs of the molecular clock

are very diverse physicochemical cues acting as synchronizers rang-
ing from heat pulses in culture medium to the sudden exposure of
cells to high serum concentration over 2 h (the term ‘‘synchronizer’’
is interchangeable with other terms, such as ‘‘entraining agent,’’
‘‘time signal,’’ or ‘‘Zeitgeber,’’ built on the German Zeit [time] and
geber [giver]). A single application of the synchronizer can reinduce
the expression of clock genes in a cell population but with a rapid
dampening effect (Balsalobre et al., 1998; Yamazaki et al., 2000;
Hirota et al., 2002), and the application of the synchronizer is sup-
posed to be repeated with a circadian periodicity to entrain the
oscillator system of cultured cells. The duration of application of a
synchronizer depends on the entrainment capacity of the oscillator
(i.e., its intrinsic period and distribution around 24 h) and is now
considered to be between 1 and 2 h after in vivo experiments on
the circadian wheel-running activity rhythm of hamsters presented
with single or double light pulses (Best et al., 1999) and in vitro
experiments on SCN neurons (Asai et al., 2001). The conditions of
resetting the phase curve responses of each cell in a culture to
external synchronizers depend on the cellular phenotype. As dem-
onstrated by modeling synchronization and rhythmicity of the cir-
cadian clock, stable synchronization depends on the synchronizer
strength (its amplitude); if it is too weak, there is no synchroniza-
tion, and if it is too strong, the system loses its synchronization
(Roenneberg and Merrow, 2002). Circadian clocks are all working
on slightly different periods close to but not exactly 24 h, giving

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rise to an asynchronous cellular populations that could be resyn-
chronized by an environmental cue (Balsalobre et al., 1998). The
temporal recording of gene expression in single cells before and
after treatment with serum-rich medium has been tried by Asai et
al. (2001) by following the fate of a single SCN cell in culture
fluorescently tagged. Similar work on fluorescently tagged Rat-1 and
primary fibroblasts (Welsh et al., 2004) or NIH3T3 cells (Nagoshi
et al., 2004) has demonstrated at the single cell level that each
fibroblast retain its own circadian rhythmicity over days in culture
and that the dampening of peripheral oscillators may be due to
desynchronization and not to a less robust clockwork inherent to
peripheral cells. The following sections will present the physico-
chemical parameters acting as synchronizers and relevant to routine
tissue culture.

Physical synchronizers: Light. By preparing primary cell cultures

from antenna, leg, or brain of a fly (Drosophila), cells are obtained
with clock genes oscillating under an environmental light cue
(Krishnan et al., 1999), but peripheral circadian photoreception has
not been demonstrated in mammals (Pando et al., 2002). Light is
the main in vivo synchronizer, but it has been relatively little ex-
plored in vitro except in studies conducted to detect a direct in-
activation of melatonin synthesis by light. Primary cultures or pineal
glands of 1-d-old neonates held in culture for 7 or 9 d synthesized
melatonin after a stimulation by norepinephrine, but under these
conditions of tissue cultures, the melatonin synthesis was sup-
pressed by light, demonstrating that the pineal cells had become
photosensitive while in culture (Tosini et al., 2000). These experi-
ments demonstrate that tissue culture conditions can drastically al-
ter some basic cellular properties: pineal cells of all nonmammalian
vertebrates are photoreceptive, whereas those of mammals do not
normally respond to light (Tosini et al., 2000). The relationship
between the central circadian clock and the light-regulated synthe-
sis of the melatonin hormone is still unresolved (Stehle et al., 2003).
As melatonin binds to members of the retinoid acid–related orphan
nuclear receptor family (ROR-ALPHA/RZR-ALPHA; Hazlerigg et
al., 1996), a molecular connection between this hormone and the
circadian clock mechanism has been suggested by Seron-Ferre´ et
al. (2002). In vitro, melatonin receptors are widely distributed in
all cell lineages, including some immortal cell lines like SCN2.2
cells (Rivera-Bermu´dez et al., 2004), allowing the design of exper-
iments to explore the connection between melatonin and the cir-
cadian clockwork.

Physical synchronizers: Temperature. The influence of heat pulses

has been explored in vitro on neurons of the suprachiasmatic nu-
cleus (Ruby et al., 1999), and two recent works have explored the
temperature compensation mechanism on peripheral cell cultures.
Circadian gene expression could be induced by a sudden increased
of 4

8

C applied to cells in culture with a thermic entrainment re-

corded by applying the sequence 12 h/37

8

C and 12 h/33

8

C (Brown

et al., 2002; Fig. 1A), and these authors have shown that by apply-
ing temperature rhythms observed in vivo to Rat-1 fibroblasts, the
circadian rhythmicity could not be induced, but entrainment on in
vivo rhythms of preexisting rhythmicity was possible. Phase shifting
of clock by heat pulses has been demonstrated in vitro on NIH-3T3
cells displaying a capacity of temperature compensation over the
temperature range of 33–42

8

C and a reinduction of circadian os-

cillations after a heat treatment at 42

8

C (Tsuchiya et al., 2003).

The NIH3T3 cells placed at 42

8

C were found to express circadian

oscillations of period2, period3, cryptochrome 1, and dbp, suggest-

ing that the induction is realized by mechanisms different from
those of the TPA or serum shock, suggesting that the circadian
rhythmicity entrained by temperature shift may be different than
those entrained by light or nutritional means (Tsuchiya et al., 2003).

In conclusion, the family of proteins containing a PAS motif that

include the main components of the circadian clock (CLOCK/
NAPS2, BMAL1, PERIOD1–3, CRYPTOCHROME1–2) is hetero-
geneous, but all these molecules display a common tridimensional
structure. They are acting as molecular sensors from bacteria to
mammals to a wide variety of cues ranging from oxygen and nitric
oxide to voltage (Dioum et al., 2002). The molecular equivalent to
CLOCK, NPAS2, has been described as a gas-responsive transcrip-
tion factor (Dioum et al., 2002). No evidence of an inactivation of
NPAS2 by oxygen or NO has been found, but carbon monoxide
could occur at the intracellular level at concentrations high enough
to interfere directly with NPAS2: BMAL-1 dimers binding on the
DNA molecules.

Chemical synchronizers: A serum shock reinduces molecular oscil-

lations of clock genes. Historically, the first demonstration was seen
with newborn and adult sera of different mammals by Balsalobre et
al. (1998): high concentration of horse serum applied for 2 h in-
duced period1, period2, rev-erb-alpha, dbp, and tef oscillation for
3 d in serum-free medium with 22.5 h of period length on Rat-1
fibroblasts, but only weak oscillations have been found on H-35
hepatoma cells. A serum shock consists of exposing a confluent cell
monolayer to a medium supplemented with 50% serum for 2 h and
measuring the reinduction of the molecular oscillations of clock
compounds, as shown in Figure 1A, for the evolution of period2
mRNA in Caco-2 cell monolayers and confirmed by many experi-
menters worldwide. An exposure to a high concentration of serum
has been related to a sharp immediate early gene response con-
cerning some factors, like the serum response factor (Chai and Tar-
nawski, 2002), and the implications of serum responsive elements
and of CLOCK:BMAL1 dimers fixation in the regulation of period1
gene have been demonstrated by Jung et al. (2003). On human
neuronal cell lines transfected with the period1 gene linked to a
luciferase reporter gene, the effect of concentrations of serum has
been explored between 20 and 40% and found to induce a lower
amplitude of luminescence variation than a single medium ex-
change with a 3-h phase delay in the first peak independent from
the dose of fetal calf serum (Motzkus et al., 2000, 2002; Maronde
and Motzkus, 2003).

Serum shock results in a rapid surge of expression of the clock

gene period1 similar to that observed in the SCN of animals re-
ceiving a light pulse (Jung et al., 2003). The rapid induction of
period1 transcription does not require any protein synthesis and is
accomplished by direct signaling pathways. The CLOCK:BMAL1
heterodimer and its cis-acting element on the promoter are involved
in serum induction of murine period1 (Jung et al., 2003).

Many blood compounds have been tested as timing ligands as

well as various substances able to trigger an immediate early gene
response. The promoter of period1 contains multiple specific re-
sponse elements for different signaling pathways, which may be
used by the circadian system to integrate different environmental
time-setting cues. Glucocorticoids (like cortisol [starvation hormone]
and receptors to glucocorticoids) are coupling signaling agents be-
tween central and peripheral oscillators in mice (Le Minh et al.,
2001). A 1-h exposure to dexamethasone, an artificial glucocorti-
coid, reinduced period1 mRNA oscillations in Rat1 fibroblasts (Bal-

317

CLOCK GENES OF MAMMALIAN CELLS

salobre et al., 2000b). Forskolin, dibutyryryl cAMP, phorbol-12-
myristate, calcimycin (A23187), epidermal growth factor, insulin,
and fibroblast growth factor applied for 1 h elicited Period1 ex-
pression (Balsalobre et al., 2000a). A wide range of products from
growth factors and interleukin-6 (Motzkus et al., 2000, 2002) to
intracellular calcium (Akashi and Nishida, 2000) as well as haem
(Kaasik and Lee, 2004) have been found to synchronize clock genes
after an application in serum-free medium for 2 h. From all these
experiments, it can be inferred that the circadian gene expressions
are autonomously controlled among the different tissues. The reg-
ulation of peripheral clocks has been also related to beta-carotene,
which is of alimentary origin, through the synthesis of the nuclear
receptor ligand retinoic acid and also the visual photopigment ret-
inol, which is produced from all trans-retinal out of dietary beta-
carotene, probably following a circadian rhythm, as the two main
enzymes (retinol dehydrogenase-7, retinal short-chain dehydroge-
nase/reductase-1) are regulated according such biological rhythms
(Panda et al., 2002). The NPAS2 exhibits a robust rhythm in the
vasculature, and retinoic acid can phase shift period2 mRNA rhyth-
micity in vivo and in serum-induced smooth muscle cells in vitro
(McNamara et al., 2001). The NPAS2 promotes E-box activation of
genes such as period1 and vasopressin and is negatively regulated
by CRY1 and CRY2 (McNamara et al., 2001). The resetting mech-
anism is different between the direct interaction of nuclear hormone
receptors with the C-terminal part of CLOCK and BMAL1 dimers
and the one driven by the glucocorticoid dexamethasone. The hu-
moral mechanism to reset a peripheral clock implicated both
CLOCK and NPAS2 by nuclear hormone receptors like angiotensin
II in the vasculature (McNamara et al., 2001; Nonaka et al., 2002)
and histone acetyl transferase in the primary cell culture of human
vascular vein (Curtis et al., 2004).

In summary, all in vitro modeling cannot be performed with few

cell lines of stromal origin (rat-1 or NIH/3T3 cell lines) as each
tissue or organ (and probably every cell type) has its own circadian
rhythmicity as well as its own sensitivities to a wide range of en-
vironmental cues.

Chemical synchronizers: Medium exchange and

D

-glucose rein-

duce molecular oscillations of clock genes. In practical cell culture,
the composition of maintenance or growth media as well as the
frequency of medium renewal are the easiest parameters to ratio-
nalize for routine control or for time-lapse experiments. A single
growth medium renewal has been found sufficient by Hirota et al.
(2002) to reinduce molecular oscillations of clock components. They
have found that the main nutrient of tissue culture media,

D

-glu-

cose, is suppose to direct cell metabolism through

D

-glucose–re-

sponsive elements that are involved in the immediate-early gene
response. In contrast to these data, Maronde and Motzkus (2003)
have reported on cell lines of neural origin transfected with period1
that a single medium renewal is insufficient to reinduce a circadian
rhythmicity. New studies on the expression of clock genes by dif-
ferent cellular phenotypes cultured in chemically defined media are
deeply needed to clarify this discrepancy.

In the isolation of fragile cells for primary cultures or to establish

new cell lines, empirical rules implying tissue sampling at different
times to maximize the recovery of quiescent or proliferative cells
should become better understood by taking into account the tissular
circadian rhythm. Some reliable methodology to obtain routinely
primary cell cultures of a highly dynamic cellular system like the
intestinal epithelial cells is still deeply needed (Fig. 1C). There are

two strategies to isolate intestinal epithelial cells. The first is pre-
serving the cell–cell adhesion and contacts between the epithelial
cells and the stromal cells to promote reinduction of the founder
cell multiplication by preserving fundamental contacts. The alter-
native is to isolate by chelation a pure population of epithelial cells
and to select single pluripotent cells in an extracellular matrice to
assay their potentials for reconstructing an epithelial sheet (Kaeffer,
2002). The design of new devices mimicking more closely the body
circadian rhythm by manipulating thermic rhythms and gas oscil-
lations according to the regulation of period genes would be useful
to reassess classic conditions to isolate and culture primary intes-
tinal cells (Fig. 1C). However, the existence of a circadian clock in
intestinal epithelial cells has been explored only recently (Pardini
et al., 2003, 2005) although intestinal circadian rhythms were
known in cell proliferation, migration, differentiation, and death
(Scheving et al., 2000). The detection of some clock genes in human
small intestine has been mentioned by Miyazaki et al. (2004), and
the tridimensional distribution of the main clock components has
been extensively studied by confocal imaging (Pardini et al., 2005).
Our results suggest that cells in proliferation–differentiation of the
human colonic crypt have retained a coordinated expression pattern
of clock components. Future work should elucidate whether this
coordination could be mediated by small molecules of metabolites
like

D

-glucose or NAD/NADP(H) acting as synchronizers and cir-

culating from bottom to top of the crypt through gap junctions or
by paracrine growth factors, like the transforming growth factor al-
pha. The transforming growth factor alpha and its main ligands, the
epidermal growth factor receptors, are both expressed along the gut,
playing a putative role in the paracrine regulation of the intestinal
crypts. The role of transforming growth factor alpha as a humoral
signal to synchronize the central circadian clock in the neurons of
the suprachiasmatic nuclei has been demonstrated by Kramer et al.
(2001). It is important to establish the connections between the
intestinal biological clock and the timing of epithelial cell-cycle
events (Bjarnason and Jordan, 2002) to improve colon cancer che-
motherapy (Le´vi, 2002) and to study the influence of food intake
on gut physiology and consumer health (Challet et al., 2003; Schi-
bler et al., 2003). Feeding cycles in transgenic rats (Stokkan et al.,
2001) or mice (Damiola et al., 2000) can entrain the clock of liver
independently of the suprachiasmatic nuclei, and according to Da-
vidson et al. (2003), the gastrointestinal system is a possible site
for a main peripheral clock.

P

ERSPECTIVES

Preserving the correct functioning of clock genes is not a pre-

requisite to success in establishing new cell lines or primary cul-
tures because the expression of these genes is rapidly dampened
after explantation without any apparent damage to the surviving
cultured cells. Nevertheless, the aim of any tissue culture model
(histotypic, organotypic, tissue reconstruction) is to reconstitute tis-
sue or organ, mimicking as closely as possible their in vivo coun-
terparts. In that respect, studies on clock genes will help us improve
our understanding of the cellular adaptation to artificial conditions
of culture and produce tissues as close as possible to the current
concept of homeostasis.

More broadly, the role of clock genes in human physiology is

progressively unraveled by relating behavioral and physiological
trends to some genomic mutation. Mutation in h-PERIOD2 phos-

318

KAEFFER AND PARDINI

phorylation sites by replacement of a serine with a glycin has been
related to the familial advanced sleep-phase syndrome (Toh et al.,
2001). Period-3 gene has been also related to delayed sleep-phase
syndrome and normal variations of morning larks and night owl
phenotypes in humans (Ebisawa et al., 2001; Archer et al., 2003),
and a clock polymorphism has been associated with human diurnal
preference (Katzenberg et al., 1998). Irregular circadian cycles,
such as night-shift work in humans (Davis et al., 2001; Hansen,
2001) or constant exposure to light in rodents, increase mammary
tumorigenesis (Anderson et al., 2000). Night or shift workers are
now representing around 20% of the workforce in our societies (Ra-
jaratnam and Arendt, 2001), and the pernicious consequences for
health of circadian rhythm alterations constitute new avenues of
research for in vitro biology.

A

CKNOWLEDGMENTS

Dr. Lissia Pardini was a recipient of a Ph.D. grant (Contract B4015 be-

tween the region Pays de la Loire, France, EU, and two INRA departments,
Biome´trie and Nasa).

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