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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 down-regulated 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 {alpha} 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.

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; PPAR{alpha}, peroxisome proliferator activated receptor {alpha}; TNF{alpha}, tumour necrosis factor {alpha}.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 ß-oxidation of fatty acids (3). The nuclear hormone receptor PPAR{alpha} (peroxisome proliferator activated receptor {alpha}) mediates transcriptional regulation of the genes associated with a biological response to peroxisome proliferators (4,5). Most hepatocytes are normally quiescent in 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 (68). There is compelling evidence that the pro-inflammatory cytokine tumour necrosis factor {alpha} (TNF{alpha}) is necessary for peroxisome proliferators to induce entry into S phase (912).

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 (1820). 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 (2224). 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 (2527). FaO cells express PPAR{alpha} 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Nafenopin (2-methyl-2-[p-( 1,2,3,4-tetrahydro-1-napthyl)-phenoxyl] propionic acid) was a gift from Ciba-Geigy (Switzerland) and TNF{alpha} 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 anti-cyclin 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 2AGoGo). In Figure 2BGo, DNA replication was assayed after 12 h of [3H]Thy incorporation. DNA replication data represent the mean (± standard deviation) of three independent experiments with two data points per experiment.



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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 + low serum) or 10% FCS (post-block + 10% FCS). DNA replication was measured by incorporation of [3H]Thy for 30 h and normalised per µg of protein.

 


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Fig. 2. (A) G1-arrested FaO hepatoma cells re-enter the cell cycle upon stimulation with nafenopin or TNF{alpha}. 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 TNF{alpha} (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

 
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 ice-cold 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 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 phase of the cell cycle. High levels of DNA replication could still be detected in 0.1% FCS after 24 h (Figure 1Go), 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 + 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 + 10% FCS), DNA replication increased by 14-fold. 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 TNF{alpha} 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 2AGo). 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 TNF{alpha} by entering S phase. DNA replication was stimulated 4-fold with nafenopin (Figure 2AGo), mimicking the response observed in primary hepatocyte cultures (8). In addition, TNF{alpha} stimulated DNA replication by 12-fold (Figure 2AGo), 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 2BGo), 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 3Go shows a complete set of data and indicates a typical experiment. CDK4 and CDK2 were present in the control, proliferating cultures (Figure 3Go, lane P) and also in arrested culture conditions (Figure 3Go, 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 G1-arrested population. However, the slower migrating (active) 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 down-regulation of the CDK inhibitor p27KIP1 protein in the arrested culture conditions compared with proliferating cells.



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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.

 
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 3Go) in all treatment groups examined (None, EGF, DMF, Naf and FCS). However, expression was greatest after FCS treatment. CDK2 and cyclin E proteins were down-regulated upon release from sodium butyrate in all treatment groups (Figure 3Go). 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 3Go).

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 3Go). 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 3Go). 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 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 (2527). 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 (3032) 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 TNF{alpha}. In this respect, the FaO model reproduces the response of cultured primary hepatocyte to non-genotoxic hepatocarcinogens. The G1-arrested 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 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 down-regulated 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 non-genotoxic 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.


    Acknowledgments
 
We thank Drs I.Kimber, S.Hasmall, S.Cosulich and S.Cariou for helpful comments during the course of this work.


    Notes
 
1 To whom correspondence should be addressed Email: stephan.chevalier{at}ctl.zeneca.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 19, 1999; revised April 8, 1999; accepted April 8, 1999.