Stimulation of DNA synthesis and c-fos mRNA expression in primary rat hepatocytes by estrogens
Chow H. Lee2 and
Anthony M. Edwards1
Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada and
1 Department of Medical Biochemistry, School of Medicine, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia
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Abstract
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The mechanism(s) of tumour promotion in liver by estrogens is not well understood although growth stimulation is known to be one important element of their action. As a basis for studying mechanisms of growth control by estrogens, effects of both natural and synthetic estrogens on DNA synthesis and protooncogene c-fos mRNA expression were examined in primary cultures of normal rat hepatocytes. 17ß-Estradiol (E2) alone was stimulatory and exhibited dramatic synergism with epidermal growth factor (EGF) in stimulating DNA synthesis. All estrogens tested (natural, synthetic, steroidal and non-steroidal) exhibited an ability to stimulate hepatocyte DNA synthesis. This appears to correlate with their ability to induce c-fos mRNA expression. In contrast to a non-estrogenic liver tumour promoter, phenobarbital, insulin is not permissive for the growth-stimulatory action of E2. Dexamethasone, which is required for stimulation of DNA synthesis by the non-estrogenic tumour promoter
-hexachlorocyclohexane and tetradecanoylphorbol acetate, completely blocked E2-stimulated DNA synthesis. Such differential requirements for auxiliary factors suggests that estrogen and other non-estrogenic liver tumour promoters act via distinct mechanisms in stimulating hepatocyte DNA synthesis. E2 alone had no effect, but when in combination with EGF significantly induced c-fos mRNA expression at early times in culture (maximal at 10 h in culture). Such findings, coupled with the observations that (i) E2 and EGF were synergistic in growth stimulation, (ii) estrogen receptor levels are higher at early times in culture and (iii) the growth-stimulatory ability of E2 is limited to 424 h in culture, support the notion that in hepatocytes E2 acts via the estrogen receptor to transactivate c-fos expression (an interaction with EGF), which ultimately culminates in enhanced DNA synthesis. Dexamethasone did not block E2-induced c-fos gene expression, suggesting that it acts in a pathway(s) distal to activation of fos gene expression. The possible inhibitory mechanisms of action of dexamethasone on E2-stimulated DNA synthesis are discussed.
Abbreviations: DES, diethylstilbestrol; E1, estrone; E2, 17ß-estradiol; E3, estriol; EE2, 17
-ethinylestradiol; EGF, epidermal growth factor; GAPDH, glyeraldehyde 3-phosphate dehydrogenase; HCH,
-hexachlorocyclohexane; PB, phenobarbital; TPA, tetradecanoylphorbol acetate.
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Introduction
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Estrogens, synthetic and endogenous, or their metabolites have been implicated in the development of various human cancers, particularly in traditional hormone-responsive target organs such as the breast, ovary and endometrium (1,2). Estrogens have also been associated with cancers in tissues which are not commonly hormone responsive, such as the liver (3). In fact, benign hepatic adenoma (46), focal nodular hyperplasia (7,8) and hepatocellular carcinoma (911) have all been linked to the use of estrogenic oral contraceptives.
In vivo experimental studies in rats and mice have confirmed the tumour-promoting ability of synthetic estrogens in hepatic neoplasia. The estrogens used in oral contraceptives, estradiol 17-phenylpropionate, estradiol benzoate, 17
-ethinylestradiol (EE2), mestranol and diethylstilbestrol (DES), have been shown to increase the size and number of liver
-glutamyltranspeptidase-positive foci and hyperplastic nodules and increase the tumour incidence in rodents following an initiating dose of diethylnitrosamine (1215). However, there are also reports on initiating activity of estrogens at relatively high doses (3,16).
There have been numerous reports supporting a role of estrogens in hepatocyte growth both in vivo and in vitro. For instance, a rise in serum 17ß-estradiol (E2) following partial hepatectomy has been observed (17,18). Exogenous estrogens administered to normal rats in vivo have been shown to stimulate DNA synthesis in hepatocytes (1922) and this may involve both direct and indirect effects via release into serum of a liver growth-stimulatory factor (23). Direct effects of estrogens, resulting in stimulation of DNA synthesis, on primary female rat hepatocytes have also been reported (2426). Estrogens alone were found to be weakly mitogenic but markedly enhanced the response of hepatocytes to other growth factors (2426). Estrogens thus appear to share with a variety of other liver tumour promoters, such as phenobarbital (PB),
-hexachlorocyclohexane (HCH), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane, pregnenolone-16
-carbonitrile and cyproterone acetate, the ability to stimulate hepatocyte growth both in vitro and in vivo (27,28). However, the observation that in rats in vivo estrogens differ from many other liver promoters, in that they do not induce monooxygenase activities, provides one indication that estrogens act on hepatocytes via a mechanism distinct from other liver tumour promoters (20).
In the traditional estrogen-sensitive cells estrogen is believed to stimulate cell proliferation and other estrogenic activity through binding to the classical estrogen receptor followed by activation of protooncogene expression (29). For instance, c-fos, c-jun and c-myc protooncogene expression was found to increase in rat uterus following administration of estrogen to ovariectomized rats (30,31). Apart from two studies which indicated that c-fos and c-myc mRNA levels in liver are not affected by either E2 or DES treatment alone (30,32), there is limited information on whether protooncogene expression is influenced by steroids in hepatocytes both in vivo and in vitro.
The present study was undertaken to evaluate in some detail the growth-stimulatory actions of estrogens in primary rat hepatocytes and their possible mechanisms of action. Our specific aims were: to determine to what extent other hormones and growth factors influence growth-stimulatory effects of estrogens; to define culture conditions for an optimal response to estrogens; to compare requirements for stimulation of DNA synthesis by other liver tumour promoters; to explore possible structureactivity relationships of natural and synthetic estrogens; to study the effects of estrogens on c-fos mRNA expression to determine its possible correlation with stimulation of DNA synthesis.
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Materials and methods
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Animals and materials
Male wistar rats (Porton strain), weighing ~200 g and given free access to laboratory chow and water, were used for hepatocyte preparation in all experiments. Williams' Medium E (WME) was obtained from Flow Laboratories (Irvine, UK), Swim's S-77 medium and antibiotics were from Gibco (Grand Island, NY) and LHSA medium was prepared as described by Oliver et al. (33). Collagenase was from Boehringer Mannheim (Penzberg, Germany), HCH was from Serva Feinbiochemica (Heidelberg, Germany) or Aldrich (Milwaukee, WI) and PB or its sodium salt were from Prosanna Laboratories (Carole Park, Queensland, Australia). All other chemicals were from Sigma (St Louis, MO). Type I collagen was extracted from rat tails essentially as described by Michalopoulos and Pitot (34). Culture dishes were collagen-coated using a solution of 0.3 mg collagen/ml 0.1% acetic acid. Stock solutions of test agents were prepared weekly. The final concentration of dimethylsulfoxide when added to cultures was 0.2% or less and had no effect on DNA synthesis and c-fos mRNA expression.
Hepatocyte isolation and culture conditions
Hepatocytes were isolated using a two-step procedure as previously described (35). Low centrifuge speeds (50 g) were used during washing of cells with LHSA medium to minimize contamination of hepatocytes with smaller non-parenchymal cells. The washed hepatocyte pellet was resuspended in warm WME containing antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin sulphate), 300 nM insulin and 3% fetal bovine serum. For DNA synthesis experiments cells were plated on 50 mm collagen-coated dishes at a density of ~3.54x104 cells/cm2. Higher cell densities, as indicated below, were used for other types of experiments. Cells were allowed to attach for 34 h at 37°C in a humidified atmosphere of 5% CO2 in air. After the attachment period cells were maintained in fresh WME (serum-free) containing antibiotics as above. This is termed `standard culture medium'. Insulin (300 nM), dexamethasone (30 nM) and epidermal growth factor (EGF) (10 ng/ml) were added depending on the nature of the experiments, as indicated in each figure legend. Other test compounds were added either after attachment or with subsequent daily medium changes or both, as indicated in the figure legends.
Measurement of the rate of hepatocyte DNA synthesis
After various times in culture the medium was replaced with fresh standard medium containing 0.08 µCi/ml [methyl-3H]thymidine (20 Ci/mmol; New England Nuclear, Boston, MA) and cells were incubated for 3 h. Following extensive washing, DNA was extracted (28), with one aliquot taken for scintillation counting and another to determine DNA concentration (36). The values shown in each figure are means ± SD from groups of four dishes per treatment obtained with a single hepatocyte preparation. Results were confirmed using independent cell preparations.
Total RNA isolation and northern analysis
Cells were harvested from cultures in solution D (4 M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol) and total RNA extracted as described by Chomczynski and Sacchi (37). Concentrations of total RNA were determined spectrometrically and 20 µg were electrophoresed in 1% agarose/formaldehyde gels and transferred overnight via capillary action onto Hybond-N+ filters (Amersham Pharmacia Biotech, Little Chalfont, UK) in 10x SSC (1.5 M NaCl, 0.15 M tri-sodium citrate, pH 7.0). Ethidium bromide staining of total RNA was used to monitor equal loading of samples. Filters were baked for 2 h at 80°C and prehybridized in 50% deionized formamide, 5x SSPE (0.9 M NaCl, 0.05 M sodium phosphate, pH 7.7, 0.005 M EDTA), 5x Denhardt's and 2% SDS for 4 h at 42°C. Hybridization was performed overnight at the same temperature in 50% deionized formamide, 5x SSPE, 10% dextran sulphate, 1x Denhardt's, 2% SDS, and >2x106 c.p.m./ml 32P-labeled v-fos or glyeraldehyde 3-phosphate dehydrogenase (GAPDH) DNA probe. Filters were then subjected to two 20 min washes at room temperature in 2x SSC and 0.1% SDS, followed by one 20 min wash at 42°C in 1x SSC and 0.1% SDS. Filters were then finally washed at 60°C in 0.5x SSC, 0.1% SDS and 0.1x SSC, 0.1% SDS for 20 min each. Autoradiography was performed using intensifying screens at 70°C and densitometric scanning was performed using an Ultroscan-XL densitometer (LKB, Rockville, MD).
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Results
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Stimulation of DNA synthesis by E2: synergism with EGF
The endogenous estrogen E2 was first tested for effects on hepatocyte DNA synthesis over a period of time, either alone or in combination with EGF. Results are shown in Figure 1
. Non-treated cultures (none) showed no evidence of cells progressing into S phase of the cell cycle. E2 alone caused a small but significant stimulation of DNA synthesis, with a peak at 40 h in culture (2.8-fold over control). At 10 ng/ml EGF was far more potent in stimulating DNA synthesis (4.3-fold over control after 40 h in culture) than E2. A synergistic effect was observed when both E2 and EGF were present in culture, with a maximal stimulation of 16-fold compared with the control at the peak of DNA synthesis. Since the optimal response of hepatocyte DNA synthesis to E2 occurred in the presence of EGF, for most experiments described below EGF was added to all cultures after the initial attachment period of 4 h. It should be mentioned here that other studies in this laboratory have shown that at the level of incorporation observed in EGF + E2-treated cultures in Figure 1
there were substantial numbers of hepatocytes (>50%) undergoing DNA synthesis, as determined by autoradiography (28).
Studies from this laboratory had previously shown that the presence of insulin throughout experiments has a permissive role in the stimulatory effects of some xenobiotics (28). The ability of xenobiotics either alone or in combination with EGF was also found to be minimal at high cell density (28). Therefore, it was of interest to investigate the effects of insulin and cell density on E2-stimulated DNA synthesis. Our results suggest that neither cell density nor insulin has any major effect on the extent of stimulation by estrogen, although this was only examined at a single time point near the peak of DNA synthesis (data not shown).
Stimulation of DNA synthesis by a wide range of estrogens
A wide range of estrogens, natural, synthetic and non-steroidal, were tested for growth-stimulatory effects on hepatocytes. The culture conditions employed (presence of EGF and measurement of DNA synthesis after 40 h in culture) were based on earlier findings for optimal stimulation by E2. Figure 2
shows the concentration dependence of the response of hepatocyte DNA synthesis to various estrogens in the range 0.330 µM. The natural estrogens [E2 > estrone (E1) > estriol (E3)] were among the most potent stimulators. The oral contraceptive steroids mestranol and EE2 have been well documented as strong promoters of hepatocarcinogenesis in rats (13,14,38). Under the present culture conditions both mestranol and EE2 had stimulatory effects on rat hepatocyte DNA synthesis, with optimal responses at 10 and 3 µM, respectively (Figure 2
). DES, a liver tumour promoter (39), and chlorotrianisene are non-steroidal estrogens and both were found to stimulate [3H]thymidine incorporation into DNA at optimal concentrations of 10 and 1 µM, respectively (Figure 2
). The effects of various concentrations (0.330 µM) of other estrogens on the growth of hepatocytes in culture were also examined. The rates of DNA synthesis observed (after 40 h in culture with an optimal concentration of each estrogen) expressed relative to rates in control dishes (EGF alone) are summarized in Table I
. The stimulations caused by the natural estrogens E1, E2 and E3 with 17ß-hydroxy or 17-keto groups at the highest concentrations tested were greater than the maximal effects of the synthetic estrogens. At relatively high concentrations various synthetic estrogens were toxic, causing cells to detach, resulting in decreasing rates of DNA synthesis as seen in Figure 2
. If compared at a lower concentration (e.g. 1 µM) all estrogens tested in Figure 2
were about equally effective in stimulating DNA synthesis (~2-fold). Of the estrogens tested, comparison of relative effects at lower concentrations suggested that d-equilenin and the non-steroidal compounds chlorotrianisene, dienestrol and hexestrol were somewhat less stimulatory than other estrogens (Table I
).
Inhibition of E2-stimulated DNA synthesis by dexamethasone
Dexamethasone has been shown to improve survival of adult hepatocytes in primary culture (40) and is routinely used in this laboratory in culture media. A permissive requirement for dexamethasone in the stimulatory effects of some tumour promoters on hepatocytes has been reported (28,41). Figure 3
shows an experiment in which effects on DNA synthesis of E2 and the classical liver tumour promoter PB were directly compared in cultures supplemented with different combinations of dexamethasone and insulin. In agreement with previous findings (28), PB required the presence of insulin for stimulation, but its effect was independent of dexamethasone. In contrast, E2 action did not require insulin and was completely abolished in the presence of dexamethasone (Figure 3
). Consistent with previous findings (28), HCH and tetradecanoylphorbol acetate (TPA) absolutely require dexamethasone for stimulation of DNA synthesis (data not shown) but repeated experiments indicated an inhibitory effect of the glucocorticoid on E2-stimulated DNA synthesis.

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Fig. 3. Differential requirement for stimulation of hepatocyte DNA synthesis by estrogen and PB. Hepatocytes were isolated and plated as described in Materials and methods. Various combinations of compounds were added after 4 h cell attachment as indicated and cultures were maintained for 40 h, when rates of DNA synthesis were measured. The additions were 30 nM dexamethasone, 300 nM insulin, 2 mM PB, 30 µM E2. Each bar is the mean ± SD (n = 4). Where treatment resulted in a mean value significantly different (P < 0.001) from corresponding control dishes (no PB or E2) this is shown by an asterisk.
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The effect of time of addition of E2 and dexamethasone on hepatocyte growth were next examined. Results are shown in Table II
. In all cultures maintained with EGF addition of E2 at 4 h only was at least as effective as adding E2 at both 4 and 24 h, whereas E2 added at 24 h resulted in only slight stimulation. This suggests that the ability of E2 to stimulate DNA synthesis may be limited either to early culture times or to early steps in the cell cycle. In the absence of E2 dexamethasone caused stimulation of DNA synthesis in EGF-treated cultures, which appeared to be mainly due to the presence of the glucocorticoid on day 1 (i.e. from 4 h). In combination with E2 through the day 2 culture period the net effect of dexamethasone was to block the strong stimulatory action of E2 and this appeared to be mainly due to the presence of dexamethasone on day 2 (i.e. from 24 h).
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Table II. Relative effects of early, late or continuous addition of dexamethasone and E2 on hepatocyte DNA synthesis
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Role of the estrogen receptor in the growth-stimulatory effect of E2
A possible role of the estrogen receptor in the mechanism of stimulation of hepatocyte DNA synthesis by E2 was investigated. The use of anti-estrogens and measurement of levels of estrogen receptor were used to assess the role of the estrogen receptor. The anti-estrogens tamoxifen and clomifene citrate were tested on the stimulatory action of E2 in cultured hepatocytes. Surprisingly, at low concentrations tamoxifen (110 µM) acted synergistically with E2 and mestranol in stimulating [3H]thymidine incorporation into DNA (data not shown). At the highest concentration tested (30 µM) tamoxifen apparently had toxic effects, causing cells to detach after 2 days in culture. The various concentrations of clomifene citrate (130 µM) tested had no effect on either E2- or mestranol-stimulated DNA synthesis (data not shown). Earlier studies by another group (42) showed that E2-stimulated hepatocyte DNA synthesis can be inhibited by tamoxifen when normal rat serum was present. In our hands addition of tamoxifen in the presence of 5% normal rat serum had no effect on E2-stimulated DNA synthesis (data not shown). To further determine the possible role of the hepatic estrogen receptor in E2 action an attempt was made to measure both the cytosolic and nuclear estrogen receptor in cultured hepatocytes, in the presence and absence of E2, under conditions where maximal stimulation of DNA synthesis can be achieved. Levels of cytosolic and nuclear estrogen receptor were determined by analysis of specific [3H]estradiol binding. Estrogen receptor content in control cultures was found to decline with time to undetectable levels after 24 h. However, binding experiments on cytosolic fractions of E2-treated cultures after 24 and 48 h resulted in linear Scatchard plots (data not shown), which showed high affinity binding with Kd values of 0.4 and 0.1 nM, respectively. The binding capacity of cultured E2-treated cells after 24 h was 7.4 fmol/mg protein and after 48 h was 3.0 fmol/mg protein. As in control cultures, the system employed showed no evidence of nuclear association of receptors, despite the presence of substantial amounts of cell extract in the assay (2 mg protein).
Role of prolactin in the growth-stimulatory effect of E2
Prolactin has been suggested to be a liver tumour promoter (43) and its concentration in serum has been shown to increase following partial hepatectomy (44). Furthermore, E2 is known to stimulate prolactin synthesis in cultured rat pituitary cells (45). It was thus of interest to investigate whether prolactin exerted a mitogenic effect on cultured hepatocytes and to examine a possible link between prolactin and the growth-stimulatory effects of E2.
As shown in Figure 4
, prolactin enhanced hepatocyte DNA synthesis dose-dependently in the absence of dexamethasone. The minimum concentration of prolactin used (20 mU/ml or 645 ng/ml) significantly stimulated DNA synthesis (P < 0.01), which is consistent with a report by Enat et al. (46). However, stimulation of DNA synthesis by prolactin was not seen when dexamethasone was included in the culture medium (Figure 4
). This effect of dexamethasone on growth stimulation by prolactin paralleled its effect on E2-stimulated DNA synthesis. To test whether E2 caused synthesis of prolactin in hepatocytes as a possible mediator of the growth stimulus by E2, levels of prolactin in medium and cells were measured 5 h after E2 addition using radioimmunoassay (kindly performed by Drs J.O.Willoughby and J.Oliver, Department of Neuroendocrinology, Flinders Medical Centre). Results indicated that prolactin was barely detectable (data not shown) in both medium and cultured hepatocytes (<1 ng/ml).

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Fig. 4. Effect of prolactin on hepatocyte DNA synthesis in the presence or absence of dexamethasone. Hepatocytes were plated as described in Materials and methods and maintained after 4 h cell attachment on `standard culture medium' with 10 ng/ml EGF, 300 nM insulin and various concentrations of prolactin as indicated. Half the cultures had 30 nM dexamethasone (). Rates of DNA synthesis were measured after 40 h in culture. Each value is the mean ± SD (n = 4). Where treatment resulted in a mean value significantly different from corresponding control dishes (no prolactin) ( ) this is shown by asterisks: *P < 0.001; **P < 0.01.
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Induction of c-fos gene expression by estrogens
E2 has been shown to induce c-fos expression in estrogen-responsive cells (30,47). In the light of this and to determine whether there is a correlation between E2-stimulated DNA synthesis and protooncogene expression, regulation of c-fos expression by estrogens in cultured hepatocytes was examined. Hepatocytes were cultured in `standard culture medium' without (none) or with EGF alone, E2 alone or EGF + E2. In each case total RNA was isolated after 1 h. Autoradiographs of northern blots hybridized with v-fos and GAPDH probes are shown in Figure 5B
and relative levels of c-fos mRNA as determined by densitometer scanning of the autoradiograph are shown in Figure 5C
. NIH 3T3 cells treated with 15% FCS for 30 min served as a positive control for c-fos expression. As shown in Figure 5
, freshly isolated hepatocytes showed undetectable levels of c-fos expression. Cells were initially unresponsive to EGF, but EGF treatment caused induction of c-fos mRNA when added after 6, 10, 34, 43 or 48 h in culture. The most significant effects of EGF treatment were seen on the second day in culture, with c-fos mRNA levels about twice background absorbance seen in the control (Figure 5
). E2 alone had no effect on c-fos expression, with a possible slight effect after 10 h in culture. EGF and E2 act synergistically to increase c-fos mRNA levels, with a peak after 10 h in culture. Synergistic induction by the EGF + E2 combination gradually decreased to a sustained lower level of expression (Figure 5C
). Identical results were obtained using cultured hepatocytes from a different cell preparation.


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Fig. 5. Effect of time in culture on c-fos inducibility. Hepatocytes were isolated and plated as described in Materials and methods. After various times in culture as shown, medium was changed to fresh `standard culture medium' with or without (none) test agents and cells were further incubated for 1 h, after which total RNA was extracted. The medium was also changed at 24 h in cultures maintained for longer periods prior to addition of test agents. Concentrations of test agents used were: 10 ng/ml EGF, 30 µM E2. Aliquots of 20 µg total RNA from hepatocytes and NIH 3T3 cells were analyzed by northern blot using v-fos and rat GAPDH cDNA probes as described in Materials and methods. Gels stained with ethidium bromide are shown in (A) and autoradiographs of the blots after hybridization are presented in (B). c-fos mRNA bands were scanned with a densitometer and the values expressed relative to absorbance of a corresponding region of the autoradiograph in the control lane for the corresponding time point (essentially equal to background in these lanes) (C).
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Since dexamethasone has been shown to completely block E2-stimulated DNA synthesis (Figure 3
), which is in contrast to HCH, for which dexamethasone is absolutely required (28), experiments were designed to compare the effects of HCH and E2 on c-fos expression under conditions where DNA synthesis can be stimulated. Figure 6
shows that without dexamethasone pretreatment, 1 h exposure of cultures to EGF, E2, HCH or EGF + HCH did not result in detectable c-fos mRNA levels, although mRNA was clearly detectable with EGF + E2 treatment under the same conditions. In cells pretreated with dexamethasone for 6 h treatment with EGF alone resulted in significant c-fos mRNA induction. While E2 and HCH alone again had no effect on c-fos expression, E2 when in combination with EGF surprisingly caused a dramatic induction of c-fos mRNA. In contrast, HCH did not affect EGF-induced c-fos expression (Figure 6
).

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Fig. 6. Effect of E2 and HCH on c-fos expression with or without dexamethasone pretreatment. Hepatocytes were isolated and plated as in Materials and methods. After 4 h for cell attachment the medium was changed to `standard culture medium' with or without 30 nM dexamethasone as shown. After 10 h in culture the medium was changed again to fresh `standard culture medium' with or without other additions as shown. Total RNA was extracted 1 h later and 20 µg total RNA from each treatment was analyzed by northern blot using v-fos DNA probe, as described in Materials and methods. (A) Equal loading of RNA samples was confirmed by ethidium bromide staining of the gel. (B) Autoradiograph of the northern blot. (C) Variations in c-fos mRNA signals from (B) were quantified by densitometric scanning and absorbance expressed in relative units. Concentrations of test agents were 10 ng/ml EGF, 30 µM E2, 30 µM HCH, 30 nM dexamethasone.
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A range of E2 concentrations was tested in combination with EGF for effects on c-fos expression. E2 concentrations of 0.33 µM had no effect, while 10 µM caused a significant increase over the EGF control (data not shown) and 30 µM caused a marked increase, as seen consistently above. Other estrogens known to have liver tumour-promoting activity were also tested for effects on c-fos expression. The results are shown in Figure 7
. In this autoradiograph the level of mRNA from EGF-treated cells is unclear because of non-specific labelling in this region of the filter. It is clear that the effects of EE2, DES and mestranol, at concentrations optimal for stimulation of DNA synthesis (Figure 2
), were much less effective in combination with EGF than E2 in inducing fos expression. These estrogens had no effect alone (Figure 7
). A replicate experiment (data not shown) also suggested that in combination with EGF the same concentrations of mestranol, DES and EE2 as in Figure 7
had much weaker effects than E2 on fos expression. When tested in combination with EGF the anti-estrogen tamoxifen (30 µM) had no effect, but it synergistically enhanced the effects of EGF + E2 on fos expression (data not shown).

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Fig. 7. Effects of other liver tumour promoter estrogens on hepatic c-fos expression in culture. Hepatocyte cultures were established and maintained for 10 h as in Figure 6 . Test agents as indicated were added with a medium change and, after 1 h, total RNA was extracted from cultures. Aliquots of 20 µg total RNA was subjected to northern analysis as described in Materials and methods. Equal loading of RNA was confirmed by ethidium bromide staining, as shown in (A). Autoradiograph of a membrane hybridized with 32P-labeled v-fos cDNA probe is shown in (B). Positions of hybridizing bands in relation to 18S and 28S are indicated. Concentrations of test agents used were 10 ng/ml EGF, 30 µM E2, 10 µM mestranol, 3 µM EE2, 10 µM DES.
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Discussion
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The results presented show that E2 alone elicits a small stimulation of DNA synthesis in cultured male rat hepatocytes, but dramatic synergism was seen in combination with EGF (Figure 1
). Studies on the time of addition of E2 show that stimulation of DNA synthesis by the steroid is primarily due to its effect at earlier times in culture (424 h), rather than later times (2440 h). Observations made here are in good agreement with findings by others (24,25). These authors found similar synergistic effects between EE2 and EGF or transforming growth factor
on female hepatocyte DNA synthesis. In their studies EE2 was present from 4 to 22 h in culture, then EGF from 22 to 34 h in culture. In contrast to our results and others (24,25) there is one report which showed that E2 dose-dependently inhibits EGF-induced DNA synthesis (42). Reasons for such a discrepancy are not clear.
Our results indicate that relatively high concentrations of estrogens (130 M), an order of magnitude higher than required to saturate the estrogen receptor in some other cell types, were necessary to evoke clear DNA synthesis responses. This is consistent with other studies in hepatocytes in which high estrogen concentrations were necessary for biological responses (2426,4850). This is most likely due to the fact that estrogens are rapidly metabolized in hepatocytes (51).
The growth-stimulatory effect of E2 was not dependent on addition of insulin after cell attachment nor was it dependent on cell density and was completely blocked by dexamethasone. This overall pattern of interaction with other factors differs from other liver tumour promoters. For instance, HCH- and TPA-induced DNA synthesis was observed only in cultures with dexamethasone, while insulin had no effect (28). On the other hand, PB action required the continuous presence of insulin but was largely unaffected by dexamethasone. Thus, the differential effects of auxiliary factors on E2- and other promoter-stimulated DNA synthesis provide indirect evidence that these structurally distinct groups of compounds may influence DNA synthesis in hepatocytes via different mechanisms.
It is becoming clear that there is a strong correlation between the potency of compounds in stimulating DNA synthesis and their effects as liver tumour promoters. For instance, the relative abilities of HCH, PB, EE2, E2, mestranol and DDT to stimulate DNA synthesis in vitro parallel their relative effects as promoters in vivo (28). On the other hand, compounds which do not promote liver carcinogenesis (barbituric acid, diphenylhydantoin, saccharin, caffeine and lithocholate) have no effects on hepatocyte DNA synthesis (28). Although estrogens share with other promoters their ability to stimulate hepatocyte DNA synthesis, their mechanism of action is likely to be distinct. For instance, Ochs et al. (20) found that while estrogens share with other liver tumour promoters the ability to stimulate liver growth, they have no significant effect on hepatic monooxygenase activities. Another piece of evidence supporting the above notion comes from studies on the hepatic EGF receptor. EGF receptor levels have been reported to decrease following treatment with PB (52), peroxisomal proliferators (53) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (54). On the other hand, EE2 was found to elevate EGF receptor levels both in vivo and in vitro (22,24,49). In addition, unlike estrogens, some other liver promoters, including HCH, DDT, PB and peroxisome proliferators, had no effect on c-fos gene expression (C.H.Lee and A.M.Edwards, in preparation). Taken together, there is strong evidence that estrogens may act to stimulate liver growth via a mechanism(s) distinct from other liver tumour promoters.
At the outset of this study there was some evidence indicating that estrogen receptors are involved in stimulation of liver growth by estrogens. Liver regeneration following partial hepatectomy (17,18) and stimulation of growth by exogenous estrogens are associated with increased levels of estrogen receptor and the anti-estrogen tamoxifen has been shown to inhibit liver regeneration (42) and induction of liver DNA synthesis in vivo by EE2 and mestranol (22,38). The fact that all estrogens (steroidal and non-steroidal) tested in this study induced DNA synthesis with comparable potency would be consistent with involvement of the estrogen receptor. Several studies (55,56), including the present one, show declining levels of hepatic estrogen receptor in primary culture. Declining levels of hepatic estrogen receptor in culture may be due to absence of the hormonal milieu required to maintain estrogen receptor levels (57,58). This observation may be related to more marked effects of estrogens added early, rather than late, in culture and indicate that estrogens act at an early time point in the cell cycle (see below). While others have used tamoxifen as an anti-estrogen in vivo to investigate involvement of the estrogen receptor in liver responses to estrogens, related studies in our culture have not yielded useful information. At the relative concentrations tested tamoxifen showed no evidence of antagonizing E2 action and, in fact, enhanced it. This is consistent with some reports that tamoxifen has partial agonist activity (16) and that it can induce estrogen receptor gene expression (59).
In this study we have shown that prolactin has a growth-stimulatory effect on primary hepatocytes. We have further shown that, like E2-stimulated DNA synthesis, prolactin-stimulated DNA synthesis can be blocked by dexamethasone (Figure 4
). This suggests that growth stimulation by both agents may be linked. Although E2 is known to stimulate prolactin synthesis in cultured pituitary cells (45), it is unlikely to do so in cultured hepatocytes. We found that levels of prolactin in the medium and cells were low (<1 ng/ml) and unlikely to be growth-stimulatory in hepatocytes. Thus, prolactin is unlikely to play an autocrine role in liver, although it is possible that in vivo stimulation of prolactin release from non-hepatocytes might have some role in the overall effects of estrogens on the liver. Prolactin has been shown to increase growth-related gene expression (c-myc and ornithine decarboxylase) and protein kinase C activity in liver (60). However, it is not known if these effects are directly related to the action of prolactin as a liver mitogen nor is it known if prolactin receptors (61,62) are involved in the present study.
The present study shows that E2 alone is ineffective in influencing c-fos mRNA expression in cultured hepatocytes. No significant induction could be seen at any of the culture times tested (Figure 5
) or when the period of exposure to the steroid was up to 10 h (data not shown). We found that EGF is essentially required for E2 to induce c-fos mRNA. Such a close association with EGF could be related to the observation that estrogen can increase EGF receptor levels in cultured hepatocytes (24,49), although it is not clear whether the effects of E2 on EGF receptor levels would occur within the 1 h experimental period of our studies. In other cell types, such as fibroblasts, it is known that fos expression may be stimulated by EGF, acting by transcriptional control at the serum response element adjacent to the fos gene (63,64). If E2 action involves direct control of fos transcription (see below) it may be that synergism with EGF reflects a cooperative interaction between E2- and EGF-controlled transcription factors. Alternatively, EGF binding to its receptor is known to result in tyrosine phosphorylation of various substrates (6567). Migliaccio et al. (68) have demonstrated that tyrosine phosphorylation of an in vitro synthesized estrogen receptor can promote maximal hormone binding to the receptor. Thus it is possible that EGF-stimulated tyrosine phosphorylation of the estrogen receptor enhances its ability to stimulate fos expression.
The region upstream of the fos gene contains an estrogen-responsive element (47) and there is substantial evidence from Wiesz and co-workers that the estrogen receptor mediates E2-induced c-fos expression in the uterus in vivo (30,47). It is therefore reasonable to speculate that fos expression in hepatocytes may also be mediated by the estrogen receptor. As discussed earlier, estrogen receptor levels are initially low in hepatocyte cultures and decline with time. Thus estrogen receptor-mediated events may be limited to early times in culture. Consistent with this, stimulation of c-fos expression by E2 was observed only at earlier times (maximal at 10 h) (Figure 5
), in parallel with the greater effects on DNA synthesis of early addition of E2 to cultures (Table II
). This provides indirect evidence of a link between early fos expression and subsequent DNA synthesis. It was, however, surprising that other estrogens, at concentrations which stimulate DNA synthesis, had much less effect than E2 in inducing c-fos expression. This may reflect their lower affinity for the estrogen receptor or alternatively their higher metabolic profile in hepatocytes.
Dexamethasone has been shown to inhibit hepatocyte DNA synthesis when given in the prereplicative phase after partial hepatectomy (69). In line with in vivo observations, the present study showed that low concentrations of dexamethasone (30 nM) can block E2-stimulated DNA synthesis but have no effect on E2-induced c-fos expression. This implies that the inhibitory effect of dexamethasone must be at some point after induction of c-fos mRNA expression. In fact, there is evidence to suggest a direct interaction between Fos protein and activated glucocorticoid receptor (70) or between the FosJun complex and activated glucocorticoid receptor (71). These complexes are believed to be unable to transactivate target genes, including genes which may be involved in later stages of the cell cycle. While the short-term presence of dexamethasone with EGF ± E2 for 1 h had no effect on c-fos mRNA levels, pretreatment with the glucocorticoid for 6 h improved the `sensitivity' of c-fos induction to EGF and, in particular, to EGF + E2 (Figure 6
). Dexamethasone has been shown to increase EGF binding in cultured hepatocytes (72) and may thus increase the ability of cells to respond to EGF. Dexamethasone, in combination with other factors, has also been shown to induce estrogen receptor mRNA expression in liver cells (73,74) and may therefore sensitize hepatic c-fos expression to E2. This observation may also be the consequence of dexamethasone stabilizing a broad range of mRNAs in cultured hepatocytes (75).
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Notes
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2 To whom correspondence should be addressed Email: leec{at}unbc.ca. 
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Acknowledgments
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C.H.L. was the recipient of a Flinders University Research Scholarship. This study was supported by grants from the Anti-cancer Foundation of South Australia.
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Received March 23, 2001;
revised May 2, 2001;
accepted May 10, 2001.