Carcinogenesis vol.20 no.7 pp.1209–1213, 1999
G1-arrested FaO cells re-enter the cell cycle upon stimulation with
the rodent non-genotoxic hepatocarcinogen nafenopin
S.Chevalier1 and R.A.Roberts
Zeneca Central Toxicology Laboratory, Cancer Biology Group,
Alderley Park, Macclesfield SK10 4TJ, UK
1To whom correspondence should be addressed
Email: [email protected]
The peroxisome proliferators are rodent non-genotoxic
hepatocarcinogens that suppress apoptosis and induce DNA
replication, cell proliferation and liver tumours. In order
to investigate the effect of peroxisome proliferators on cell
cycle progression, we arrested the well-differentiated rat
hepatoma cell line FaO in the G1 phase of the cell cycle.
Under these conditions, CDK2 and CDK4 protein expression remained unchanged compared with proliferating
cells, but expression of cyclin D1 and p27KIP1 was downregulated and cyclin E accumulated in the inactive form.
G1-arrested cells were able to enter the cell cycle on addition
of exogenous growth factors such as epidermal growth
factor (EGF) or hepatocyte growth factor (HGF) and
replicate their DNA within 12 to 24 h of re-stimulation.
Upon release from G1 arrest, CDK2 protein expression was
down-regulated and, surprisingly, p27KIP1 expression was
restored. Cyclin D1 and phosphorylated cyclin E accumulated at 12 h but were degraded by 24 h after addition of
EGF. Importantly, the peroxisome proliferator nafenopin
and tumour necrosis factor α were able to induce DNA
replication. Thus, the profile of expression of cell cycle
regulatory proteins upon stimulation with nafenopin is
comparable with that induced by growth factors such
as EGF.
Introduction
Non-genotoxic carcinogens constitute a diverse group of chemicals of therapeutic, industrial and environmental significance
that can cause tumours in rodents, despite their lack of
genotoxic activity (1,2). The data available to date suggest
that non-genotoxic carcinogens cause tumours in rodents by
perturbing growth regulation in their target tissue, leading to
the induction of DNA synthesis and cell proliferation (2,3).
The peroxisome proliferator class of non-genotoxic carcinogens
causes peroxisome proliferation and liver tumours in rats
and mice, accompanied by liver enlargement and elevated
transcription of cytochrome P4504A1 and acyl-CoA oxidase,
a key enzyme in β-oxidation of fatty acids (3). The nuclear
hormone receptor PPARα (peroxisome proliferator activated
receptor α) mediates transcriptional regulation of the genes
associated with a biological response to peroxisome proliferators (4,5). Most hepatocytes are normally quiescent in
Abbreviations: CDK, cyclin-dependent kinase; CKIs, cyclin-dependent kinase
inhibitors; EGF, epidermal growth factor; FCS, fetal calf serum; HGF,
hepatocyte growth factor; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PPARα, peroxisome proliferator activated receptor
α; TNFα, tumour necrosis factor α.
© Oxford University Press
the rodent adult liver but the peroxisome proliferator nafenopin
and the barbiturate drug, phenobarbitone, induce entry into
S phase both in vivo and in primary hepatocyte cultures
(6–8). There is compelling evidence that the pro-inflammatory
cytokine tumour necrosis factor α (TNFα) is necessary for
peroxisome proliferators to induce entry into S phase (9–12).
The decision to undergo cell proliferation occurs as cells
pass a restriction point in G1, after which they become
refractory to extracellular growth signals and commit to the
autonomous programme that carries them through to division
(13). Therefore, alteration of the components that control
progression through G1 phase may permit the sustained increase
in cell proliferation associated with carcinogenesis. In fibroblast
cell lines, progression in G1 and entry into S phase are both
governed by precise regulation of the expression of cyclins,
cyclin-dependent kinases (CDKs) and cyclin-dependent kinase
inhibitors (CKIs) (14,16). The CDK4–cyclin D and CDK2–
cyclin E complexes promote progression within G1 phase and
at the G1/S phase transition, respectively (14,15). Moreover,
the kinase activity of these CDK–cyclin complexes is regulated
negatively by CKIs such as p27KIP1 and p21CIP1 (16).
The progression of primary hepatocyte through the restriction point in G1 upon stimulation with epidermal growth factor
(EGF) correlates with an increase in the expression of cyclin
D1 and E mRNA and CDK2 protein (17). Although cyclin E
protein was expressed at a constant level, its activation in late
G1 was coincident with a modification of its phosphorylation
status (17). In comparable studies of regenerating liver, cyclin
D1 and E proteins were present in resting liver and displayed
modest variation in expression throughout the cell cycle
(18–20). The activity of both CKD4–cyclin D1 and CKD2–
cyclin E was upregulated late in G1 phase (18,20,21). The
CKI, p21CIP1 was not detected in quiescent mouse liver but
protein levels increased steadily after partial hepatectomy
(20,21). In contrast, p27KIP1 was present in quiescent liver and
its expression level did not decline as hepatocytes entered the
cell cycle (20,21).
An increased expression of G1- and G2/M-associated cyclins
and CDKs in response to peroxisome proliferators has been
shown both in vivo and in vitro using primary hepatocyte
cultures (22–24). However, despite their physiological relevance, primary hepatocyte cultures can be limiting due to their
inherent complexity and short life-span (2–4 days). Therefore,
the identification of a well-differentiated hepatoma cell line
that can retain a response to peroxisome proliferators, FaO,
was a significant step forward in modelling the mechanism of
action of the peroxisome proliferators (25–27). FaO cells
express PPARα and respond to the peroxisome proliferator
nafenopin, by an induction of cytochrome p4504A1 expression
(25,26). Here we demonstrate the arrest of FaO cells in early
G1 permitting cell cycle entry upon stimulation with nafenopin
or with EGF. Furthermore, we report the protein expression
profile of cell cycle regulatory proteins involved at the G1/S
transition in this synchronized FaO cell system.
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S.Chevalier and R.A.Roberts
Fig. 1. Synchronization of the rat FaO hepatoma cell line in G1 phase of the
cell cycle. DNA replication was measured in proliferating conditions
(control) and after 24 h in 0.1% FCS (low serum). After the double block
treatment, DNA replication was measured upon re-addition of either 0.1%
FCS (post-block 1 low serum) or 10% FCS (post-block 1 10% FCS).
DNA replication was measured by incorporation of [3H]Thy for 30 h and
normalised per µg of protein.
Materials and methods
Chemicals
Nafenopin (2-methyl-2-[p-( 1,2,3,4-tetrahydro-1-napthyl)-phenoxyl] propionic
acid) was a gift from Ciba-Geigy (Switzerland) and TNFα was from Insight
Biotechnology (UK). Sodium butyrate, EGF, hepatocyte growth factor (HGF)
and insulin were from Sigma (UK). Ham F12 medium and fetal calf serum
(FCS) were purchased from Advanced Protein Products (UK). Primary anticyclin D1, anti-cyclin E, anti-CDK4 and anti-CDK2 antibodies were purchased
from Santa Cruz Biotechnology (UK) and anti-p27KIP1 antibodies from
Transduction Laboratories (UK). Secondary antibodies and enhanced chemiluminescence detection reagent were from Pierce (UK). Redivue [methyl-3H]thymidine ([3H]Thy: 25 Ci/mmol) was purchased from Amersham Life
Science (UK).
Cell culture and G1 arrest
The FaO cell line derived from the Reuber H35 rat hepatoma was obtained
from Dr M.Weiss (Institut Pasteur, Paris, France) (28) and subcultured in
Ham F12 supplemented with penicillin (100 U/ml), streptomycin (100 g/ml),
L-glutamine (2 mM) and 10% FCS at 37°C in humidified atmosphere of 5%
CO2/95% air. Cells were placed in flasks at a density of 80 000 cells/cm2 for
24 h before medium changing. G1 arrest of the FaO cells was achieved by a
24 h period in 0.1% FCS followed by addition of 10 mM sodium butyrate
for a further 24 h. Treated cells were washed twice with Ham F12 medium
and restimulated for 30 h in the presence of [3H]Thy before assaying for
DNA replication (Figures 1 and 2A). In Figure 2B, DNA replication was
assayed after 12 h of [3H]Thy incorporation. DNA replication data represent
the mean (6 standard deviation) of three independent experiments with two
data points per experiment.
DNA replication assay
[3H]Thy was incubated (0.5 µl/ml culture medium) for the time period
indicated in the figure legends. After incubation, cells were washed twice in
warm (37°C) phosphate-buffered saline (PBS) and scraped from the flasks in
ice-cold PBS. Aliquots were taken for protein concentration (Bio-Rad, UK)
and DNA from the cell lysate was precipitated with 30% trichloroacetic acid
(TCA, Sigma, UK) on a GF/C filter (Whatman, UK) and washed twice with
5% TCA using a Manifold system (Millipore, UK). [3H]Thy incorporation
was counted on a liquid scintillation analyser (Packard, UK) using Optiphase
‘Hisafe’3 scintillation liquid (Wallac, UK) and corrected for 1 µg of protein.
Western blotting
The protein samples were prepared by scraping cells from the flasks into icecold PBS. Samples were then sonicated and centrifuged in EDTA (2 mM)
and phenylmethylsulfonyl fluoride (PMSF, 2 mM). The protein content of the
supernatant was calculated using the BioRad protein assay. SDS polyacrylamide gels were prepared by standard techniques. A 20 µg sample of protein
was loaded per lane onto a 12.5% acrylamide gel. Protein was blotted onto a
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Fig. 2. (A) G1-arrested FaO hepatoma cells re-enter the cell cycle upon
stimulation with nafenopin or TNFα. FaO cells arrested in G1 phase were
able to re-enter the cell cycle upon stimulation with 10% FCS, or growth
factors such as EGF (25 ng/ml), HGF (2 ng/ml), insulin (10 ng/ml) or
nafenopin (20 µM) or TNFα (25 ng/ml). DNA replication was measured by
incorporation of [3H]Thy for 30 h and normalised per µg of protein. (B)
Synchronized FaO cells enter S phase within 12 h. DNA replication in FaO
cells arrested in G1 and re-stimulated in the cell cycle with 10% FCS was
measured by pulse labelling of [3H]Thy for the indicated time and
normalised per µg of protein
PVDF filter (Immobilon-P, Millipore, UK) and probed using primary then
secondary antibodies and detected using enhanced chemiluminescence and
Hyperfilm ECL (Amersham Life Science, UK). Equal protein loading on the
PVDF filter was verified using Ponceau S staining (Sigma, UK).
Results
Synchronization of the rat FaO hepatoma cell line in the G1
phase of the cell cycle
In order to permit measurement of cell cycle entry, we arrested
the well-differentiated FaO rat hepatoma cell line in the G1
Nafenopin-induced S phase in G1-synchronized FaO cells
phase of the cell cycle. High levels of DNA replication could
still be detected in 0.1% FCS after 24 h (Figure 1), or even
after 48 h (data not shown). In order to achieve a complete
arrest of the FaO cells in G1, a ‘double block’ was required
using serum starvation (0.1% FCS) for 48 h and sodium
butyrate treatment for the final 24 h. When sodium butyrate
was removed from the culture medium, DNA replication
assayed after 30 h was reduced by 10-fold (post-block 1 low
serum), compared with control (10% FCS). Therefore, there
was no stimulation in S phase upon release from G1 arrest.
When sodium butyrate was removed from the culture medium
and FaO cells were re-stimulated with 10% FCS for 30 h
(post-block 1 10% FCS), DNA replication increased by 14fold. This strong induction suggests that cells re-entered the
cell cycle synchronously. Thus, the double block treatment
synchronised differentiated FaO rat hepatoma cell line in the
G1 phase of the cell cycle to mediate a reversible growth arrest.
The non-genotoxic carcinogen nafenopin or TNFα stimulate
arrested FaO cells to enter the cell cycle
To determine if arrested FaO could respond to defined growth
factors in serum free conditions, arrested FaO cells were
stimulated with EGF or HGF at a concentration shown to
induce rat hepatocytes S phase (25 and 2 ng/ml, respectively)
(29 and data not shown) or with insulin (10 ng/ml) (Figure
2A). Under these conditions, there was an 8-fold stimulation
with EGF and insulin and a 5-fold stimulation with HGF,
indicating that arrested FaO cells retain the capacity to respond
to growth factors and that the synchronization protocol was
not deleterious.
Next, we determined if arrested FaO cells could respond to
the peroxisome proliferator nafenopin and to TNFα by entering
S phase. DNA replication was stimulated 4-fold with nafenopin
(Figure 2A), mimicking the response observed in primary
hepatocyte cultures (8). In addition, TNFα stimulated DNA
replication by 12-fold (Figure 2A), a stronger induction than
that noted previously in primary hepatocytes (12). In order to
investigate changes in protein expression when cells progress
in G1 phase, we determined the time course of S phase entry
using pulse labelling experiments. The majority of [3H]Thy
incorporation was between 12 and 24 h post stimulation with
FCS (Figure 2B), indicating that the cells re-enter S phase and
replicate their DNA starting at 12 h after stimulation. These
synchronized FaO cells stopped incorporating [3H]Thy at 24 h,
suggesting progression into G2 phase of the cell cycle. Although
the levels of stimulation of DNA replication with EGF or
nafenopin were lower than with FCS, they both induced a
similar time course pattern of [3H]Thy incorporation when
compared with FCS (data not shown).
Butyrate-mediated G1 arrest inhibits p27KIP1 protein expression
and induces accumulation of dephosphorylated cyclin E
Having established that FaO cells can be arrested in G1, then
restimulated to enter the cell cycle by FCS, EGF and nafenopin,
we studied the molecular events that underpin these changes.
Figure 3 shows a complete set of data and indicates a typical
experiment. CDK4 and CDK2 were present in the control,
proliferating cultures (Figure 3, lane P) and also in arrested
culture conditions (Figure 3, lane G1). On the other hand,
cyclin D1 protein was expressed in proliferating cells but, as
predicted, its expression was decreased in the G1-arrested
population. Several cyclin E migrating forms were detected in
proliferating cells but net expression was increased in the G1arrested population. However, the slower migrating (active)
Fig. 3. Western blotting analysis of cell cycle regulatory proteins in
synchronized FaO cells. Total cell lysates from proliferating cells (P) or
G1-arrested cells (G1) were analysed by western blotting with the indicated
primary antibodies. Upon release from G1 arrest, cells were treated with no
addition (None), EGF, DMF, nafenopin or 10% FCS for 12 or 24 h and
total cell lysates were analysed by western blotting.
form predominated in control proliferating cells and the two
faster migrating forms of cyclin E (inactive) were overexpressed in G1-arrested cells. Surprisingly, we observed a downregulation of the CDK inhibitor p27KIP1 protein in the arrested
culture conditions compared with proliferating cells.
The non-genotoxic carcinogen nafenopin induced a similar
pattern of cyclin expression to the growth factor EGF
Having determined the pattern of expression of cell cycle
regulating proteins in control proliferating versus G1-arrested
cells, we wished to determine how nafenopin may alter
expression to drive cell cycle entry. In addition, we wanted to
determine the response to FCS and EGF, a potent hepatic
mitogen. Expression of cell cycle regulating proteins was
analysed by western blotting at 12 h (end of G1 phase) and at
24 h (end of S phase) after release from G1 arrest.
Relative to the down-regulation induced by sodium butyrate,
p27KIP1 expression was restored at 12 h after release from G1
arrest (Figure 3) in all treatment groups examined (None, EGF,
DMF, Naf and FCS). However, expression was greatest after
FCS treatment. CDK2 and cyclin E proteins were downregulated upon release from sodium butyrate in all treatment
groups (Figure 3). In addition, the inactive, fast migrating
form of cyclin E completely disappeared whereas the slower
migrating, active form remained present at both 12 and 24 h.
As expected, the expression of CDK4 remained constant
whereas cyclin D1 protein was induced at 12 h after release
from G1 arrest (Figure 3).
Upon stimulation with FCS or EGF, CDK2, CDK4 and
p27KIP1 protein expression appeared to be unchanged compared
with the cells released from G1 arrest but not stimulated
(Figure 3). Interestingly, cyclin D1 was expressed at 12 h upon
stimulation with FCS or EGF but was completely degraded
by 24 h at the point when cells exit S phase (Figure 3). Cyclin
E protein expression was comparable with unstimulated control
at 12 h after EGF but the majority was degraded by 24 h,
reflecting the progression of arrested FaO cells through S
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S.Chevalier and R.A.Roberts
phase. A similar pattern of cyclin E expression was seen with
FCS, although some was still visible at 24 h.
Surprisingly, the nafenopin solvent control DMF (dimethylformamide) appeared to induce some changes in protein
expression in synchronized FaO cells, despite the finding that
it did not stimulate cells to enter S phase. However, upon
stimulation of the arrested FaO cells with nafenopin, the
changes in protein expression seen were very similar to those
induced by EGF. In particular, nafenopin shared the ability of
EGF and FCS to induce cyclin D1 expression at 12 h followed
by degradation at 24 h.
Discussion
Peroxisome proliferators induce hepatocyte proliferation and
hepatocarcinogenesis in rat and mouse (2,3). To study growth
changes in response to peroxisome proliferators, we evaluated
cell cycle entry and its regulation in the well-differentiated rat
hepatoma cell line, FaO, since these cells have conserved
some ability to respond to peroxisome proliferators (25–27).
Previously, the stimulation of cell proliferation by peroxisome
proliferators in FaO cells has not been demonstrated, perhaps
because they were at their maximal rate of cell proliferation.
Since most hepatocytes are quiescent in the adult rodent liver
and cultured hepatocytes do not divide in vitro, the lack of a
demonstration of S-phase entry in FaO could also be due to
the inherent asynchrony of cell lines. Hence, it was necessary
to halt proliferation in these cells to determine the extent of
stimulation by individual effectors. Therefore, we established
a protocol to permit arrest of the FaO hepatoma cell line in
early G1 phase. Sodium butyrate has been shown to arrest
fibroblasts and hepatoma cell lines in early G1 phase of the
cell cycle (30–32) and to cause accumulation in G1 of normal
rat kidney fibroblasts (33). Sodium butyrate inhibits cyclin D1
and c-myc gene expression (34,35), stimulates the expression
of the CKI, p21CIP1 and blocks phosphorylation of the retinoblastoma protein (pRb) in mouse fibroblasts (36). Here, we
have shown that treatment of FaO cells with sodium butyrate
mediated a reversible growth arrest since arrested cells were
able to re-enter the cell cycle upon stimulation with defined
growth factors such as EGF, HGF or insulin. More importantly,
FaO cells arrested in G1 were able to re-enter the cell cycle
upon stimulation with nafenopin or TNFα. In this respect, the
FaO model reproduces the response of cultured primary
hepatocyte to non-genotoxic hepatocarcinogens. The G1arrested FaO cells entered S phase 12 h after stimulation,
providing a synchronized, timed model for analysis of key cell
cycle regulatory proteins. The expression of CDK2 and cyclin
E proteins before and after arrest in G1 phase correlates with
their expression pattern in studies of regenerating liver and
primary hepatocytes (17,18,20). The accumulation of the fast
migrating form of cyclin E in arrested FaO cells suggests
inactivity of the CKD2–cyclin E complex in G1. Cyclin D1
protein expression was reduced after G1 arrest compared with
levels in proliferating FaO cells. This correlates with a decrease
in cyclin D1 mRNA level observed in response to sodium
butyrate in fibroblasts (34). Overall, in response to sodium
butyrate treatment, FaO cells appear to overexpress an inactive
form of cyclin E and down-regulate cyclin D1; the net result
of these changes would be to inhibit the CDK phosphorylation
activity required for progression in S phase.
It is perhaps more surprising that the CKI, p27KIP1, was
expressed in proliferating but not in G1-arrested FaO since a
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decrease in p27KIP1 expression is generally associated with
cell proliferation in fibroblast cultures (16). However, p27KIP1
was expressed in resting liver (20), in unstimulated rat primary
hepatocytes that are naturally arrested in the G1 phase of the
cell cycle and in proliferating primary hepatocytes (S.Chevalier,
in preparation). Furthermore, in primary culture of dog thyroid
epithelial cells, progression in S phase was fully compatible
with expression of p27KIP1 (37). One explanation is that in
some cell systems, including hepatocytes and FaO, there may
be high levels of p27KIP1 protein, but sequestered in an inactive
form. Taken together, the data presented indicate that the
arrested FaO system reproduces the protein expression patterns
of cell cycle regulatory proteins described in other systems.
Our data suggest that the regulation of induction of S phase
with nafenopin did not differ dramatically from the induction
of S phase by a defined growth factor such as EGF or by FCS.
Specifically, CDK2, CDK4 and p27KIP1 expression levels were
similar and cyclin D1 and cyclin E expression were downregulated at 24 h when cells exit S phase after stimulation
with FCS, EGF or nafenopin. Despite a lack of induction of
S phase with the DMF solvent, there were some changes in
the expression of these cell cycle regulatory proteins, probably
due to the ability of low concentrations of DMF to induce cell
survival as reported in other rat cell lines (38). Alternative
solvents for nafenopin such as ethanol or dimethylsulfoxide
(DMSO) could be used but these carry their own limitations;
DMSO is a strong inducer of hepatocyte differentiation and
survival (39) whereas ethanol causes both apoptotic cell death
and cell proliferation (40).
There is a need to understand how chemicals induce nongenotoxic carcinogenesis to facilitate early identification of
the potential for neoplasia. Here we have demonstrated that
the well-differentiated FaO cell line can undergo a synchronized
entry into S phase in response to the peroxisome proliferator,
nafenopin, providing an opportunity to investigate the molecular regulation of cell cycle entry. The data provide evidence
for a role for cyclin E and D1, but suggest that the markers
examined are common to the S-phase response to growth
factors. In summary, we have characterised at the molecular
level the S-phase entry response of FaO to peroxisome proliferators and shown that this is not associated with gross
dysregulation of CDK or cyclin expression.
Acknowledgements
We thank Drs I.Kimber, S.Hasmall, S.Cosulich and S.Cariou for helpful
comments during the course of this work.
References
1. Ashby,J. and Tennant,R.W. (1991) Definitive relationships among chemical
structure, carcinogenicity and mutagenicity for 301 chemicals tested by
the U.S. NTP. Mutat. Res., 257, 229–306.
2. Chevalier,S. and Roberts,R.A. (1998) Perturbation of rodent hepatocyte
growth control by non-genotoxic hepatocarcinogens: mechanisms and lack
of relevance for human health. Oncol. Rep., 5, 1319–1327.
3. Cattley,R.C., DeLuca,J., Elcombe,C., Fenner-Crisp,P., Lake,B.G.,
Marsman,D.S., Pastoor,T.A., Popp,J.A., Robinson,D.E., Schwetz,B.,
Tugwood,J. and Wahli,W. (1998) Do peroxisome proliferating compounds
pose a hepatocarcinogenic hazard to humans? Regul. Toxicol. Pharmacol.,
27, 47–60.
4. Issemann,I. and Green,S. (1990) Activation of a member of the steroid
hormone receptor superfamily by peroxisome proliferators. Nature, 347,
645–650.
5. Lee,S.S., Pineau,T., Drago,J., Lee,E.J., Owens,J.W., Kroetz,D.L.,
Fernandez-Salguero,P.M., Westphal,H. and Gonzalez,F.J. (1995) Targeted
disruption of the alpha isoform of the peroxisome proliferator-activated
Nafenopin-induced S phase in G1-synchronized FaO cells
receptor gene in mice results in abolishment of the pleitrophic effects of
peroxisome proliferators. Mol. Cell. Biol., 15, 3012–3022.
6. Columbano,A. and Shinozuka,H. (1996) Liver regeneration versus direct
hyperplasia. FASEB J., 10, 1118–1128.
7. Roberts,R.A., Soames,A.R., Gill,J.H., James,N.H. and Wheeldon,E.B.
(1995) Non-genotoxic hepatocarcinogens stimulate DNA synthesis and
their withdrawal induces apoptosis, but in different hepatocyte populations.
Carcinogenesis, 16, 1693–1698.
8. James,N.H. and Roberts,R.A. (1996) Species differences in response to
peroxisome proliferators correlate in vitro with induction of DNA synthesis
rather than with suppression of apoptosis. Carcinogenesis, 17, 1623–1632.
9. Akerman,P., Cote,P., Yang,S.Q., McClain,C., Nelson,S., Bagby,G.J. and
Diehl,A.M. (1992) Antibodies to tumor necrosis factor-alpha inhibit liver
regeneration after partial hepatectomy. Am. J. Physiol., 263, 579–585.
10. Beyer,H.S. and Theologides,A. (1993) Tumor necrosis factor-alpha is a
direct hepatocyte mitogen in the rat. Biochem. Mol. Biol. Int., 29, 1–4.
11. Bojes,H.K., Germolec,D.R., Simeonova,P., Bruccoleri,A., Schoonhoven,R.,
Luster,M.I. and Thurman,R.G. (1997) Antibodies to tumor necrosis factoralpha prevent increases in cell replication in liver due to the potent
peroxisome proliferator WY-14,643. Carcinogenesis, 18, 669–674.
12. Rolfe,M., James,N.H. and Roberts,R.A. (1997) Tumour necrosis factor
alpha (TNF-alpha) suppresses apoptosis and induces DNA synthesis in
rodent hepatocytes: a mediator of the hepatocarcinogenicity of peroxisome
proliferators? Carcinogenesis, 18, 2277–2280.
13. Pardee,A.B. (1989) G1 events and regulation of cell proliferation. Science,
246, 603–608.
14. Bartek,J., Bartkova,J. and Lukas,J. (1997) The retinoblastoma protein
pathway in cell cycle control and cancer. Exp. Cell Res., 237, 1–6.
15. Chevalier,S. and Blow,J.J. (1996) Cell cycle control of replication initiation
in eukaryotes. Curr. Opin. Cell. Biol., 8, 815–821.
16. Sherr,C.J. and Roberts,J.M. (1995) Inhibitors of mammalian G1 cyclindependent kinases. Genes Dev., 9, 1149–1163.
17. Loyer,P., Cariou,S., Glaise,D., Bilodeau,M., Baffet,G. and GuguenGuillouzo,C. (1996) Growth factor dependence of progression through G1
and S phases of adult rat hepatocytes in vitro. J. Biol. Chem., 271,
11484–11492.
18. Loyer,P., Glaise,D., Cariou,S., Baffet,G., Meijer,L. and GuguenGuillouzo,C. (1994) Expression and activation of CDKs (1 and 2) and
cyclins in the cell cycle progression during liver regeneration. J. Biol.
Chem., 269, 2491–2500.
19. Albrecht,J.H., Hu,M.Y. and Cerra,F.B. (1995) Distinct patterns of cyclin
D1 regulation in models of liver regeneration and human liver. Biochem.
Biophys. Res. Commun., 209, 648–655.
20. Albrecht,J.H., Poon,R.Y.C., Ahonen,C.L., Rieland,B.M., Deng,C. and
Crary,G.S. (1998) Involvement of p21 and p27 in the regulation of CDK
activity and cell cycle progression in the regenerating rat liver. Oncogene,
16, 2141–2150.
21. Kato,A., Ota,S., Bamba,H., Wong,R.M., Ohmura,E., Imai,Y. and
Matsuzaki,F. (1998) Regulation of cyclin D dependent kinase activity in
rat liver regeneration. Biochem. Biophys. Res. Commun., 245, 70–74.
22. Rininger,J.A., Goldsworthy,T.L. and Babish,J.G. (1997) Time-course
comparison of cell cycle protein expression following partial hepatectomy
and Wy-14,643-induced hepatic proliferation in F344 rats. Carcinogenesis,
18, 935–941.
23. Gill,J.H., Brickell,P., Dive,C. and Roberts,R.A. (1998) The rodent nongenotoxic hepatocarcinogen nafenopin suppresses apoptosis preferentially
in non-cycling hepatocytes but also elevates CDK4, a cell cycle progression
factor. Carcinogenesis, 19, 1743–1747.
24. Peters,J.M., Aoyama,T., Cattley,R.C., Nobumitsu,U., Hashimoto,T. and
Gonzalez,F.J. (1998) Role of peroxisome proliferator-activated receptor
a in altered cell cyle regulation in mouse liver. Carcinogenesis, 19,
1989–1994.
25. Bayly,A.C., French,N.J., Dive,C. and Roberts,R.A. (1993) Non-genotoxic
hepatocarcinogenesis in vitro: the FaO hepatoma line responds to
peroxisome proliferators and retains the ability to undergo apoptosis.
J. Cell Sci., 104, 307–315.
26. Bayly,A.C., Roberts,R.A. and Dive,C. (1994) Suppression of liver cell
apoptosis in vitro by the non-genotoxic hepatocarcinogen and peroxisome
proliferator nafenopin. J. Cell Biol., 125, 197–203.
27. Passilly,P., Jannin,B., Hassell,S.J. and Latruffe,N. (1996) Human HepG2
and rat FaO hepatic-derived cell lines show different responses to
ciprofibrate, a peroxisome proliferator: analysis by flow cytometry. Exp.
Cell Res., 223, 436–442.
28. Deschatrette,J. and Weiss,M.C. (1974) Characterization of differentiated
and dedifferentiated clones from rat hepatoma. Biochimie, 56, 1603–1611.
29. Michalopoulos,G.K. (1990) Liver regeneration: molecular mechanisms of
growth control. FASEB J., 4, 176–187.
30. Van Wijk,R., Tichonicky,L. and Kruh,J. (1981) Effect of sodium butyrate
on the hepatoma cell cycle: possible use for cell synchronization. In vitro,
10, 859–862.
31. Charollais,R.H., Buquet,C. and Mester,J. (1990) Butyrate blocks the
accumulation of cdc2 mRNA in late G1 phase but inhibits both the early
and late G1 progression in chemically transformed mouse fibroblats BPA31. J. Cell. Physiol., 145, 46–52.
32. Levavasseur,F., Burbelo,P.D., Cariou,S., Lietard,J., Yamada,Y. and
Clement,B. (1995) Nuclear recruitment of A1p145 subunit of replication
factor C in the early G1 phase of the cell cycle in Faza 567 hepatoma cell
line and hepatocyte primary cultures. FEBS Lett., 363, 132–136.
33. Joensuu,T. and Mester,J. (1994) Inhibition of cell cycle progression by
sodium butyrate in normal rat kidney fibroblasts is altered by expression
of the adenovirus 5 early 1A gene. Biosci. Reports, 14, 291–300.
34. Lallemand,F., Courilleau,D., Sabbah,M., Redeuilh,G. and Mester,J. (1996)
Direct inhibition of the expression of cyclin D1 gene by sodium butyrate.
Biochem. Biophys. Res. Commun., 229, 163–169.
35. Souleimani,A. and Asselin,C. (1993) Regulation of c-myc expression by
sodium butyrate in the colon carcinoma cell line Caco-2. FEBS Lett., 326,
45–50.
36. Buquet-Fagot,C., Lallemand,F., Charollais,R. and Mester,J. (1996) Sodium
butyrate inhibits the phosphorylation of the retinoblastoma gene product
in mouse fibroblasts by a transcription-dependent mechanism. J. Cell.
Physiol., 166, 631–636.
37. Depoortere,F., Dumont,J.E. and Roger,P.P. (1996) Paradoxical accumulation
of the cyclin-dependent kinase inhibitor p27KIP1 during the cAMPdependent mitogenic stimulation of thyroid epithelial cells. J. Cell. Sci.,
109, 1759–1764.
38. Boyle,C.C. and Hickman,J.A. (1997) Toxin-induced increase in survival
factor receptors: modulation of the threshold for apoptosis. Cancer Res.,
57, 2404–2409.
39. Kost,D.P. and Michalopoulos,G.K. (1991) Effect of 2% DMSO on the
mitogenic properties of epidermal growth factor and hepatocyte growth
factor in primary hepatocyte culture. J. Cell. Physiol., 147, 274–280.
40. Baroni,G.S., Marucci,L., Benedetti,A., Mancini,R., Jezequel,A.M. and
Orlandi,F. (1994) Chronic ethanol feeding increases apoptosis and cell
proliferation in rat liver. J. Hepatol., 20, 508–513.
Received January 19, 1999; revised and accepted April 8, 1999
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