Potentiation of epidermal growth factor-induced DNA synthesis in rat hepatocytes by phenobarbitone: possible involvement of oxidative stress and kinase activation
N.J. Hodges,
T.C. Orton1,
A.J. Strain2 and
J.K. Chipman3
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT,
1 Safety of Medicines Department, AstraZeneca, Alderley Park, Macclesfield SK10 4TG and
2 Liver Research Laboratories, Queen Elizabeth Hospital, Birmingham B15 2TH, UK
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Abstract
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A transient induction of S phase DNA synthesis is a common feature of non-genotoxic rodent hepatocarcinogens when administered in vivo. In the present study the ability of phenobarbitone (PB) to induce S phase DNA synthesis in primary cultures of rat hepatocytes was investigated. In the absence of serum or growth factors PB was not a mitogen per se. However, stimulation of S phase DNA synthesis by epidermal growth factor (EGF) was enhanced by co-culture with PB. This effect was both time and concentration dependent. The lowest concentration of PB that significantly enhanced the effect of EGF was 10 µM and the effect was maximal at 1.0 mM. At a concentration of 2.0 mM PB no longer enhanced EGF-induced S phase DNA synthesis. Hepatocyte cultures pretreated with PB (0.1 mM) for 2 days were more responsive to the induction of S phase DNA synthesis by EGF for the subsequent 2 days. Despite the inhibition of PB enhancement of S phase DNA synthesis by the antioxidant dimethylthiourea, reduced glutathione was not depleted by PB treatment nor were oxidized glutathione or lipid peroxides elevated. Western blotting analysis showed that PB had no effect on epidermal growth factor receptor (EGFR) autophosphorylation per se after 1 and 48 h culture, enhanced sensitization of EGFR therefore does not appear to contribute to the enhancement of S phase DNA synthesis by PB. In contrast, treatment of hepatocytes with PB for 12 h resulted in a small but statistically significant activation of p42/44 MAP kinase activity and activation of protein kinase C, as measured by redistribution of enzyme activity from a soluble to a particulate compartment of hepatocytes. Therefore, PB-mediated changes in protein kinase activity may contribute to the potentiation this compound affords.
Abbreviations: DMSO, dimethylsulphoxide; DMTU, dimethylthiourea; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GJIC, gap junctional intercellular communication; GSH, reduced glutathione; GSSG, oxidized glutathione; HBSS, Hank's balanced salt solution; HMBA, hexamethylene bisacetamide; M6PR, mannose 6-phosphate receptor; MAP kinase, mitogen-activated protein kinase; OPT, o-phthalaldehyde; PB, phenobarbitone; PBS, phosphate-buffered saline; PKC, protein kinase C; PNH, perfluoro-n-hexane; ROS, reactive oxygen species; TGF
, transforming growth factor
; TGFß, transforming growth factor ß; TPA, 12-O-tetradecanoylphorbol 13-acetate.
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Introduction
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Phenobarbitone (PB) promotes the formation of rat liver tumours but the mechanism is poorly understood. A typical response of hepatocytes to tumour promoters in vivo is an increase in S phase DNA synthesis (1,2). In vivo PB-mediated events do not occur uniformly throughout the liver but are mainly restricted to the pericentral region of liver lobules (3). Pericentral hypertrophy is maintained throughout the exposure period to PB. In contrast, DNA synthesis is transient, peaking after 3 days and returning to basal levels after 1 week (4). The basis of the adaptive process is uncertain. In the mouse, PB (2500 p.p.m. in the diet) causes transient (<1 week) increases in transforming growth factor
(TGF
) and epidermal growth factor receptor (EGFR) and decreases in transforming growth factor ß (TGFß) and mannose 6-phosphate receptor (M6PR) (5), which all correlate with cell replication.
The transient increase in DNA synthesis may be a necessary feature of the promotional process, although there is no spatial or temporal correlation between DNA synthesis and subsequent tumour formation. Chronic PB administration (>3 months) reduces the capacity of normal hepatocytes to proliferate in response to mitogenic stimuli (58). This correlates with inhibition of two stimulating pathways of DNA synthesis: a reduction in EGFR numbers (58) and loss of the ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) to activate protein kinase C (PKC). The latter was linked to the loss of PKC translocation due to PB exposure (9). In parallel, an increase in TGFß in periportal hepatocytes has also been observed (10). It is possible that `initiated' cells are resistant to these mitoinhibitory signals, allowing their clonal expansion and hence tumour formation (1114).
In vitro PB elevates epidermal growth factor (EGF)-dependent S phase DNA synthesis but is not a mitogen per se (1517). We wished to study the basis of this synergy between PB and EGF with particular emphasis on the time dependency and mechanism. PB has been observed to enhance the formation of reactive oxygen species (ROS) in neoplastic rat liver nodules (18,19) and there is substantial evidence for a role of ROS in many of the effects of PB (2025), possibly via the uncoupling of cytochrome P450 (18,26,27). Therefore, we studied the potential intermediacy of ROS. Previous studies have shown an involvement of ROS in many models of tumour promotion (2833) which may in part be mediated by an influence on kinases in the signal transduction pathways triggered by EGF (3438). We therefore included a study of EGFR autophosphorylation and kinase activity of some of the components of the EGF-mediated signalling cascade.
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Materials and methods
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All chemicals were obtained from Sigma unless stated otherwise and were of the highest grade available.
Cell isolation and culture
Male Wistar rats (Biomedical Services Unit, University of Birmingham) weighing ~250 g were used to isolate hepatocytes by the two-step collagenase perfusion method (39). Briefly, the liver was perfused through the hepatic portal vein with 350 ml of calcium/magnesium-free Hank's balanced salt solution (HBSS). After the initial perfusion, the liver was perfused with 350 ml of HBSS containing 1.1 mg/ml collagenase A (Boehringer Mannheim) for 15 min. The isolated cells were washed three times in HBSS containing 4.0 mM calcium and finally resuspended in William's medium E supplemented with 10% fetal calf serum. The viability of preparations used in subsequent experiments as measured by trypan blue dye exclusion was >80%. Hepatocytes were plated at a density of 2.1x104 cells/cm2 in cell culture vessels (Falcon; Becton Dickinson) that had been precoated with rat tail collagen type 1 (10 µg/ml in 3.3 mM acetic acid). After an initial attachment period of ~2 h, the medium was removed and the cultures washed with phosphate-buffered saline (PBS). Fresh serum-free medium supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.0 mM glutamine, 100 nM insulin and 30 nM dexamethasone was added. All test compounds were also added at this time, which was arbitrarily designated time 0. The medium was changed every subsequent 24 h.
Measurement of S phase DNA synthesis
For autoradiography, cultures in 35 mm dishes were treated overnight with 1.0 µCi/ml [5'-3H]thymidine (Amersham). Cultures were washed with ice-cold PBS (three times) and fixed for 20 min with ice-cold methanol. Fixed plates were coated with K2 emulsion (Ilford) and stored in a dark box at 4°C. After ~1 week autoradiographs were developed with Kodak L-24 diluted 1:5 with water for 3 min and fixed with a 30% (w/v) solution of sodium thiosulphate (5 min). Labelling indices were evaluated by calculating the percentage of hepatocytes with labelled nuclei in three fields in duplicate or triplicate dishes.
Measurement of reduced glutathione (GSH) and oxidized glutathione (GSSG)
Hepatocytes (4x106) were cultured in the presence of test compounds for the appropriate length of time. The medium was removed and the cultures washed with PBS. All subsequent steps were carried out at 4°C. PBS (2 ml) was added and the cells extracted by the addition of 1 ml of extraction buffer (2.9 mM Na2EDTA in 14% perchloric acid) followed by gentle scraping with a plastic cell scraper. The mixture was placed on ice for 15 min and centrifuged for 5 min (13 000 r.p.m., bench microfuge). To 2 ml of the supernatant was added 1 ml of neutralizing buffer (1 M KOH, 1 M KHCO3) and the mixture centrifuged as above. The resulting supernatant was either assayed immediately or stored at 70°C by flash freezing in liquid nitrogen.
GSH assay
Determination of GSH was performed as described by Hissin and Hilf (40). To 100 ml of the supernatant were added 1.8 ml of phosphateEDTA buffer (0.1 M NaH2PO4·2H2O, 0.005 M Na4EDTA, pH 8.0) and 100 µl of 1 mg/ml o-phthalaldehyde (OPT). After mixing and incubation at room temperature for 15 min the solution was transferred to a quartz curvette. Fluorescence at 420 nm was determined with activation at 350 nm using a fluorimeter (Perkin Elmer Luminescence LS50B).
GSSG assay
To measure GSSG a 100 µl aliquot of the original supernatant was incubated at room temperature with 40 µl of 0.04 M N-ethylmaleimide for 30 min. To this mixture were added 1.76 ml of 0.1 M NaOH and 100 µl of 1 mg/ml OPT. The resulting mixture was incubated for 15 min at room temperature and used to assay GSSG fluorometrically as outlined above.
Measurement of lipid peroxidation
Cultured hepatocytes (4x106) were washed in ice-cold PBS (10 ml) and gently scraped into PBS (10 ml) using a plastic cell scraper. The cells were pelleted by centrifugation and resuspended in PBS (0.5 ml) and lysed by vigorous pipetting. Total lipid peroxide and malonaldehyde were estimated colourmetrically using a LPO-586 kit (Bioxytech) according to the manufacturer's instructions.
Measurement of PKC activity
Cytosolic and membrane PKC extracts were prepared using a modified version of a method described previously (34). Cultured hepatocytes (4x106) were scraped into ice-cold extraction buffer (25 mM TrisHCl, 0.5 mM EDTA, 0.5 mM EGTA, 100 µg/ml leupeptin and 0.5 mM phenylmethylsulphonyl fluoride, pH 7.4) using a plastic cell scraper. The cell suspension was lysed by ultrasound with 4x10 s bursts using the micro probe of a sonicator (Ultrasonics model no W-225; Heat Systems) and the homogenate centrifuged at 100 000 g for 1 h at 4°C. The supernatant was collected for determining cytosol-associated PKC activity. The pellet was washed in ice-cold extraction buffer and resuspended in buffer containing 0.2% (v/v) Triton X-100. The resulting suspension was incubated on ice for 45 min and then centrifuged at 100 000 g (4°C) for a further 30 min. The resulting supernatant was used to determine membrane-associated PKC activity. Samples were assayed using a Signatect Kit PKC assay kit (Promega) according to the manufacturer's instructions.
Mitogen-activated protein kinase (MAP kinase) assay
Extracts were prepared by scraping hepatocytes into RIPA buffer [0.6% NP-40, 150 mM NaCl, 10 mM HEPES, pH 7.9, 1 mM EDTA, 1 tablet of complete protease inhibitor (Boehringer) per 50 ml of buffer]. The suspension was placed on ice for 20 min with occasional vortexing and centrifuged at 15 000 g (20 min, 4°C). The supernatant was decanted into a fresh 1.5 ml Eppendorf tube and either assayed straight away or stored at 80°C. MAP kinase activity was measured using a Biotrak p42/44 MAP kinase enzyme assay system (Amersham) according to the manufacturer's instructions.
Western blotting
After exposure to test compounds, cultured hepatocytes (6x105) were scraped into 1 ml of sodium vanadate buffer (PBS containing 10 mM EDTA, 50 mM NaF, 10 mM Na3VO4, pH 7.4). The cells were centrifuged at 13 000 r.p.m. for 2 min, the supernatant removed and the pellet homogenized in 400 µl of vanadate buffer. The process was repeated and the final cell homogenate used for western blotting. The hepatocyte homogenates were mixed with an equal volume of Laemmeli loading buffer and the proteins separated under reducing conditions by SDSPAGE using an 8% polyacrylamide gel and electroblotted onto a nitrocellulose membrane (0.2 µm; Bio-Rad). The membrane was subjected to immunodetection using the PT66 anti-phosphotyrosine antibody (P-3300; Sigma) followed by peroxidase-conjugated goat anti-mouse secondary antibody (A-9917; Sigma). Visualization was carried out using ECL (Amersham). Membranes were stained with Ponceau red to confirm equal protein loading.
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Results
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Effect of PB on background and EGF-induced S phase
In the absence of serum, PB (1.0 mM) was not a mitogen in primary cultures of rat hepatocytes. However the stimulation of S phase DNA synthesis by EGF (Figure 1
) was elevated by co-incubation with PB. The lowest concentration of PB that significantly enhanced EGF-induced S phase DNA synthesis was 10 µM, the response was biphasic with respect to concentration and maximal at 1.0 mM (Figure 2
). The apparent lag period between PB exposure and subsequent PB enhancement (Figure 1
) suggests a role for PB priming. The presence of PB for the final day of a 3 day treatment with EGF was not sufficient for S phase enhancement (data not shown). This suggests that the lag period is not entirely a result of the delay between exposure of cultures to EGF and the subsequent stimulation of S phase DNA synthesis. Further evidence for the priming effect of PB is shown in Figure 3
. Cultures primed with 0.1 mM PB (2 days) were significantly more responsive to the mitogenic effect of EGF for 2 days after priming.

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Fig. 1. Time dependency of PB enhancement of EGF-induced S phase DNA synthesis. [3H]thymidine (1 µCi/ml) was present in the medium for the final 16 h of culture. The labelling index (%LI) was calculated following autoradiography. ***, Significantly different from control (P < 0.001, ANOVA). , Significantly different from EGF alone (P < 0.05, ANOVA). For comparative purposes the similar enhancing effect of PNH (0.1 mM) on EGF-induced S phase is shown. The results represent the means ± SD of three experiments carried out in duplicate (n = 3).
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Fig. 2. Concentration dependency of PB enhancement of EGF-induced S phase DNA synthesis. Hepatocytes were cultured for 3 days. [3H]thymidine (1 µCi/ml) was present in the medium for the final 16 h of culture. The labelling index (%LI) was calculated following autoradiography. *, **, ***, Significantly different from control (P < 0.05, 0.01 and 0.001, respectively, ANOVA). The results represent the means ± SD of three experiments carried out in duplicate (n = 3).
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Relationship between the priming effect of PB and oxidative stress
A possible role for oxidants was implicated in that the antioxidant dimethylthiourea (DMTU) inhibited (43.2%) the ability of PB to enhance the effect of EGF (Table I
). Furthermore, the the ability of the P450 uncoupler perfluoro-n-hexane (PNH, 0.1 mM) to enhance EGF-induced S phase was similar to that observed with PB (Figure 1
). In contrast, the differentiating agent hexamethylenebisacetamide (HMBA) had no effect. Dimethylsulphoxide (DMSO), which acts as both a differentiating agent and an antioxidant, reduced the EGF response (64.4% inhibition) (Table I
) and therefore could not be used to study PB enhancement of the EGF response. Neither DMTU nor HMBA influenced the EGF response in the absence of PB, suggesting that the reduction in the EGF response by DMSO is not a result of its antioxidant effect. Parameters related to oxidative stress for hepatocytes treated with PB are shown in Table II
. Despite the apparent inhibition of PB-enhanced S phase DNA synthesis by the antioxidant DMTU, GSH was not depleted by PB nor were GSSG or lipid peroxides elevated (Table II
). As a positive control, treatment of cultures with 1.0 mM diethylmaleate for 1 h depleted GSH by 77.2%. Despite these negative results, the possibility that PB induces a low level of oxidative stress has not been excluded.
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Table I. Effect of 2% (v/v) DMSO, 0.5 mM HMBA and 25 mM DMTU on EGF (50 ng/ml)-induced S phase DNA synthesis and its enhancement by PB (1.0 mM) in 3 day cultures of primary rat hepatocytes
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The effect of PB on EGFR autophosphorylation
We next tested the hypothesis that the effects of PB might be mediated by the modulation of certain kinases, including autophosphorylation of EGFR. The addition of EGF to cultures for 1 h induced receptor autophosphorylation, as represented by augmentation of the 170 kDa band representing the EGFR (Figure 4
). PB did not elevate receptor autophosphorylation and had no effect on EGF-induced autophosphorylation of EGFR (Figure 4
). After 48 h continuous exposure, EGF no longer induced receptor autophosphorylation (Figure 5
). Furthermore, PB had no effect on receptor autophosphorylation at this time point either in the absence of EGF or when co-cultured with EGF (Figure 5
). When cultures were primed with 0.1 mM PB (2 days) no change in either the basal level of receptor autophosphorylation or EGFR expression was observed. Furthermore, PB-primed cultures were no more responsive to EGF-induced receptor autophosphorylation (data not shown). The findings from these western blotting experiments fit in well with our earlier observation that PB is not a mitogen per se. However, the priming ability of PB does not appear to be a result of changes in either intrinsic levels of EGFR autophosphorylation or in susceptibility to ligand (EGF)-induced receptor phosphorylation.

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Fig. 4. Western blot of phosphorylated EGFR. Hepatocytes were treated for 1 h with 1.0 mM PB or 50 ng/ml EGF in the presence or absence of PB. Phosphorylated EGFR was detected with the PT66 antibody and is apparent at ~170 kDa ( ). A representative gel from two independent experiments is shown.
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Fig. 5. Western blot of phosphorylated EGFR. Hepatocytes were treated for 48 h with 1.0 mM PB or 50 ng/ml EGF in the presence or absence of PB. Phosphorylated EGFR was detected with the PT66 antibody and is apparent at ~170 kDa ( ). As a positive control hepatocytes were treated with EGF for 1 h. A representative gel from two independent experiments is shown.
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The effect of PB on PKC activity
Activation of PKC has been operationally measured as the redistribution of activity from a soluble to a particulate fraction of cells homogenized in the presence of calcium chelators (41). In untreated hepatocytes, 32% of the total PKC activity was recovered in the particulate fraction of the cell. In contrast, after treatment of hepatocytes with the PKC activator TPA (50 nM) for 30 min 80% of the total PKC activity was recovered in the particulate fraction of the cell (Figure 6
). Interestingly, culture of hepatocytes for 12 h with PB (1.0 mM) resulted in activation of PKC with 78% of the total PKC activity being recovered in the particulate fraction. Furthermore, PB-mediated translocation of PKC activity to the particulate fraction was not further effected by subsequent treatment with TPA (Figure 6
).

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Fig. 6. Lipid-dependent cytosolic and membrane-associated PKC activity in hepatocytes cultured in the presence and absence of 1.0 mM PB for 12 h prior to addition of 50 nM TPA for 30 min. PKC activity was assessed by the transfer of 32P from [ -32P]ATP to a specific peptide substrate followed by phosphorimaging analysis. *, Significantly different from the control (P < 0.05, paired t-test). The results represent the means ± SD of three experiments carried out in duplicate (n = 3). 1.48x105 volume units are equivalent to 10 pmol ATP/min/mg protein.
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The effect of PB on MAP kinase activity
The basal level of p42/44 MAP kinase activity in control hepatocytes was 10 pmol ATP/min/mg protein. Activation of p42/44 MAP kinase activity by EGF was rapid, with a maximal 4.8-fold increase in kinase activity being observed after just 1 min of EGF treatment compared with time 0 controls (Figure 7
). After 12 h p42/44 MAP kinase activity in EGF-treated cells remained elevated and was 1.52-fold greater than that of control cells at the same time point. Although co-culture with PB had no effect on either the magnitude or time course of the EGF-induced initial transient (<1 h) activation of p42/44 MAP kinase activity, culture of hepatocytes with PB for 12 h resulted in a small but statistically significant (P < 0.05) increase in p42/44 MAP kinase activity similar in magnitude to the response to EGF (Figure 8
).

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Fig. 7. Time course of EGF activation of p42/44 MAP kinase in primary cultures of rat hepatocytes. MAP kinase activity was assessed by the transfer of 32P from [ -32P]ATP to a specific peptide substrate followed by scintillation counting. The results represent the means of two experiments carried out in duplicate.
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Fig. 8. Time course of p42/44 MAP kinase. MAP kinase activity was assessed by the transfer of 32P from [ -32P]ATP to a specific peptide substrate followed by scintillation counting. *, Significantly different from the appropriate control (P < 0.05, ANOVA). The results represent the means ± SD of three experiments carried out in duplicate (n = 3).
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Discussion
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There are multiple changes in response to PB in rodents that might contribute to the transient increase in hepatocellular proliferation which, if not attenuated as occurs in normal hepatocytes, may contribute to tumour promotion. These changes include elevated levels of EGFR and its endogenous ligand TGF
and decreases in the levels of mitoinhibitory factor TGFß and its receptor M6PR (5). The multiplicity of changes make it difficult to delineate the critical initial interactions of PB in hepatocytes responsible for this response. Although it has previously been shown that PB can enhance EGF-induced S phase DNA synthesis in primary cultures of rat hepatocytes (1517), the mechanism of this enhancement remains unknown.
We have demonstrated that, in accordance with other findings (1517), PB is not a mitogen per se in cultured hepatocytes in the absence of serum and growth factors. The synergy with EGF regarding S phase enhancement was concentration dependent and seen only between 10 µM and 1.5 mM, in agreement with previous findings (17). Interestingly, the minimum concentration of PB that resulted in a significant enhancement of EGF-induced S phase (10 µM) was less than the plasma concentration of 60 µM PB achieved in rats chronically treated with PB (0.1% in drinking water) for 3 months (42). Furthermore, in the mouse a similar dose of PB (0.25% in the diet) results in transient hepatocyte proliferation in vivo (5). Our results indicate that the response to PB exposure is time dependent. Pre-exposure experiments suggest that the synergy between EGF and PB depends on a `priming' ability of PB. To our knowledge this has not been demonstrated previously with PB in primary cultures of hepatocytes. However, a similar priming ability of the peroxisome proliferator nafenopin has been observed (43).
Various studies have demonstrated a role for ROS in the effects of PB (2025). Although our results show that GSH was not depleted nor were GSSG or lipid peroxides elevated by PB treatment, we have not excluded the possibility that PB induces low levels of oxidative stress in primary cultures of rat hepatocytes, as is evidenced by changes produced in vitro in liver by PB (25). This hypothesis is supported by our finding that DMSO and DMTU inhibit the synergy between EGF and PB. DMSO is a solvent that is a strong differentiating agent known to induce differentiation of tumour cells (44). Both DMSO and DMTU are potent scavengers of ROS and may destroy ROS formed under conventional culture conditions. In contrast to the effects of DMSO and DMTU on PB enhancement of EGF-induced S phase, we observed that HMBA, which is a differentiating agent (45) but not an antioxidant, was unable to inhibit the synergy between EGF and PB. These findings are in agreement with Kojima et al. (46), who found that in primary cultures of rat hepatocytes inhibition of gap junctional intercellular communication (GJIC), considered to be related to oxidative stress, was overcome by DMSO and DMTU but not HMBA.
Activation of protein kinases is a critical and universal response in cells to both growth and differentiation signals. There is evidence for a role of phosphorylation events in transcriptional activation by PB. For example, serine/threonine phosphorylation participates in the induction of rodent cytochrome P450 isozymes by PB. Furthermore, the PB-dependent nuclear translocation of the constitutively active receptor involved in regulation of CYP 2B genes also requires phosphorylation (47). Interestingly, the role of protein phosphorylation has also being implicated for protein binding of the phenobarbitone response element in which nuclear factor
B was implicated (48). Activation of EGFR-associated tyrosine kinase activity by ligand-dependent binding is the first step of the pathway by which the EGFR transduces signals leading to cell proliferation (49). In the present study we have demonstrated that in primary cultures of rat hepatocytes PB had no effect on either ligand (EGF)-dependent or -independent EGFR autophosphorylation at the time points investigated. Furthermore, the `priming' ability of PB was not a result of an increased sensitivity of the EGFR to subsequent EGF-induced autophosphorylation (data not shown).
MAP kinases are activated in response to a variety of extracellular signals, including growth factors, hormones and neurotransmitters that activate distinct intracellular pathways (5052). Substrates for MAP kinase include other protein kinases (e.g. c-RAF 1 and S6 kinase), phospholipase A2, cytoskeletal proteins and transcription factors (e.g. c-fos and c-jun), however, the precise role of MAP kinase activation in primary hepatocyte cultures remains poorly defined. In the present study activation of p42/44 MAP kinase by EGF was rapid with a maximal response being observed after just 1 min of EGF treatment. Studies by other workers (50) have demonstrated a similar time course of p42/44 MAP kinase activation by hepatocyte growth factor in primary hepatocyte cultures. We have shown that treatment of hepatocytes with PB (1.0 mM, 12 h) results in a statistically significant activation of p42/44 MAP kinase. To our knowledge this effect has not been demonstrated previously with PB in either primary cultures of rat hepatocytes or in vivo. The biological significance of this small and time-dependent activation is unclear. Although partial activation of p42/44 MAP kinase by PB is not sufficient to stimulate S phase DNA synthesis in primary hepatocye cultures (PB is not a mitogen per se), it may be one of the mechanisms by which PB enhances EGF-induced S phase DNA synthesis.
Detection of specific binding of the tumour promoter TPA to PKC (53) suggests that PKC can play an important role in tumour promotion. The translocation of PKC from the cytosol to the plasma membrane is known to be important for activation because pretreatment of intact cells, including hepatocytes (41), with TPA decreased the activity recovered from a subcellular soluble fraction while increasing that in the membranous fraction. In the current study we have demonstrated that in primary cultures of rat hepatocytes treatment with both PB (1.0 mM, 12 h) and TPA resulted in translocation of lipid-dependent PKC activity from a cytosolic to a membranous compartment of the cell. Jirtle and Meyer (17) have demonstrated a similar response to TPA in primary cultures of hepatocytes. They also found that PB (5.0 mM, 1 h) had no effect on the distribution of PKC within the cell but at concentrations in excess of 2 mM we failed to see enhancement of S phase DNA synthesis by PB. Redistribution of PKC to the cytoplasmic membrane allows the enzyme access to a range of protein substrates, many of which are thought to be involved in the signal transduction of factors influencing mitogenesis. Interestingly, a recent study (54) demonstrated that in hepatocytes the diacylglycerol-dependent PKC pathway is not activated by EGF, but rather is involved in mediating enhancement of responsiveness to EGF by other hormones (e.g. vasopressin and angiotensin II). Therefore, activation of PKC by PB may provide an explanation for the observed synergy between EGF and PB on S phase DNA synthesis in primary cultures of rat hepatocytes.
In contrast to the transient activation of PKC by PB in primary cultures of rat hepatocytes, a study by Brockenbrough et al. (9) showed that chronic treatment with PB in vivo had no effect on the distribution of PKC in unstimulated hepatocytes. Furthermore, hepatocytes from PB-treated rats were less sensitive to subsequent TPA activation of PKC. These findings correlate with the inhibitory effect of chronic PB exposure on the growth of normal (uninitiated) hepatocytes. Like TPA, PB may have time-dependent effects on PKC activity, with a transient activation being followed by inhibition following chronic treatment with PB in vivo. If initiated or putative preneoplastic cells become refractory to these growth-inhibitory effects as previously demonstrated (1,13), then chronic PB exposure may lead to clonal expansion of preneoplastic liver cells and to increased incidence of tumours. Another possible important factor that, combined with the above, may influence hepatocellular proliferation is the inhibition of GJIC by PB (21,23,55). Furthermore, a number of phosphorylation events have been associated with the inhibition of function of connexins involved in GJIC (56). Combined with an early reduction in TGFß in vivo, enhanced cell proliferation would ensue via a loss of mitoinhibitory influence. It is becoming increasingly apparent that generation of endogenous ROS plays an important role in EGF-mediated cell signalling (57,58). Our results suggest that one of the important influences of PB regarding cell proliferation is modulation of at least some of the components of the EGF/EGFR signalling cascade. We postulate that PB-mediated generation of low levels of ROS may be responsible for these effects.
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Notes
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3 To whom correspondence should be addressed Email: j.k.chipman{at}bham.ac.uk 
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Acknowledgments
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We thank Jane Davies and Dr James Sidaway for their technical support and advice and acknowledge financial support from the BBSRC and AstraZeneca.
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References
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Received February 11, 2000;
revised July 17, 2000;
accepted July 24, 2000.