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Carcinogenesis, Vol. 21, No. 6, 1205-1211, June 2000
© 2000 Oxford University Press

Carcinogenesis

Inhibition of TGF-ß-induced apoptosis by ethinyl estradiol in cultured, precision cut rat liver slices and hepatocytes

Jinqiang Chen, Mamata Gokhale1, Brian Schofield, Shelly Odwin and James D. Yager2

Department of Environmental Health Sciences, Division of Toxicological Sciences, The Johns Hopkins School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ethinyl estradiol (EE) is a strong promoter of hepatocarcinogenesis in the rat. Treatment with EE and other hepatic promoters induces transient growth stimulation followed by growth inhibition (mitosuppression) in hepatocytes. Previously, we identified several genes whose transcript levels were increased during EE-induced mitosuppression, including transforming growth factor ß (TGF-ß), which inhibits growth and induces apoptosis in hepatocytes. Various hepatic promoters, including phenobarbital and several peroxisomal proliferators, have been shown to inhibit TGF-ß-induced apoptosis in rat hepatocytes. The goal of this study was to investigate whether EE is also an inhibitor of TGF-ß-induced apoptosis in rat hepatocytes. Several approaches to detect apoptosis were used, including the TUNEL assay, detection of high molecular weight DNA fragmentation by field inversion gel electrophoresis and determination of cytosolic cytochrome c levels by western analysis. TGF-ß-induced apoptosis in cultured, precision cut liver slices and hepatocytes of female rats. EE (<=3 µM) completely inhibited TGF-ß-induced apoptosis in these systems in the absence of cytotoxicity. These findings add EE to the list of several hepatic promoters that both induce TGF-ß while simultaneously inhibiting its ability to cause apoptosis.

Abbreviations: EE, ethinyl estradiol; E2, estradiol; HMW, high molecular weight; ITS, 5 µg/ml insulin and transferrin and 5 ng/ml selenous acid; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; PB, phenobarbital; PBS, phosphate-buffered saline; PI, propidium iodide.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
In women, prolonged exposure (>5 years) to oral contraceptives containing ethinyl estradiol (EE) is associated with a modest increase in the risk of developing liver tumors (1). In the rat synthetic estrogens are strong promoters of hepatocarcinogenesis (reviewed in ref. 2). Effects caused by exposure to low, non-hepatotoxic doses (<=5 µg/day) of EE include a transient increase in hepatocyte growth (3–5) followed by a subsequent inhibition (mitosuppression) of basal and/or induced liver growth (6). Mitosuppression is also caused by several other hepatic tumor promoters, including phenobarbital (PB) (7–9) and the peroxisomal proliferators clofibrate, methylclofenapate and others (10,11). These mitoinhibitory effects caused by EE, PB and several peroxisome proliferators are associated with increased levels of transforming growth factor ß (TGF-ß) and/or its receptors and the mannose 6-phosphate receptor (9,12–14).

Apoptosis is a process of controlled, selective cell death involved in development, maintenance of tissue/organ homeostasis at the cellular level and elimination of cells that have experienced excessive damage. Schulte-Hermann and colleagues were among the first to study the role of apoptosis in regulating liver size and in the development of preneoplastic altered hepatic foci (15–18). These investigators identified TGF-ß as a key factor in the induction of apoptosis in hyperplastic liver undergoing regression subsequent to cessation of xenobiotic treatment (19). They also demonstrated that TGF-ß not only inhibited epidermal growth factor-induced DNA synthesis in cultured rat hepatocytes, but also caused apoptosis (20–22).

The peroxisome proliferator nafenopin and PB have been shown to inhibit TGF-ß-induced apoptosis (23–25). Subsequent studies showed that the anti-apoptotic effect of nafenopin is mediated by signaling through peroxisome proliferator receptor {alpha} (26).

As mentioned, expression of TGF-ß, which both inhibits growth and induces apoptosis in hepatocytes, is increased during EE-induced mitosuppression (13). In this study we investigated whether EE is an inhibitor of TGF-ß-induced apoptosis using cultured, precision cut rat liver slices and hepatocytes. Our results show that at low concentrations (<=3 µM) EE inhibited TGF-ß-induced apoptosis. Since apoptosis contributes to protection from carcinogenesis by selectively removing cells containing excessive or unrepaired DNA damage, spontaneous or carcinogen induced, its inhibition may represent an important mechanism by which several liver tumor promoters work.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Preparation and culture of rat hepatocytes and liver slices
Fischer 344 rats (200–300 g) were used in this study under protocols approved by the Animal Care and Use Committee. Hepatocytes were prepared from female rats by collagenase perfusion and cultured on collagen-coated dishes in Chee's medium (Gibco BRL Life Technologies, Gaithersburg, MD) supplemented with 2 mM glutamine, 1 mM arginine, 41.3 µM thymidine, 26.2 mM sodium bicarbonate (all from Sigma Chemical Co., St Louis, MO), ITS premix (5 µg/ml insulin and transferrin and 5 ng/ml selenous acid; Collaborative Research, Waltham, MA) and 50 µg/ml gentamicin, as described previously (27). Incubation was at 37°C in 5% CO2, 95% air. After allowing 2 h for attachment, the medium was changed to Chee's medium additionally supplemented with dexamethasone (1 µM) plus 1% dimethylsulfoxide. The medium was changed again 16–18 h later, at which time the treatments indicated were started. Precision cut slices were prepared from male and female rat livers. No gender differences in the responses analyzed were detected. The livers were excised, cored and used for cutting 200–250 µm thick slices in ice-cold RPMI medium using a Krumedieck tissue slicer as described previously (28). Slices were cultured in RPMI medium (Gibco BRL) supplemented with 5% fetal bovine serum (Gibco BRL), 0.5 mM L-methionine, 0.1 mM hydrocortisone hemisuccinate, 0.5 mM nicotinamide, 0.25 mg/ml hemoglobin (all from Sigma) and 1 µl/ml ITS. Slices were floated onto inserts and placed in glass vials containing 4 ml of medium. Caps with holes in the center were put on the vials to allow continuous oxygenation while the slices were rolled inside a dynamic roller incubator at 37°C in an atmosphere of 95% O2, 5% CO2. The medium was changed after an initial 2 h culture period. Treatment was started at this time and continued for 24 or 48 h, as indicated.

Microscopic detection of apoptosis
For fluorescence microscopy, the hepatocytes were fixed in 10% buffered formalin for 2 h and stained using the Hoechst dye 33258 (Sigma). The TUNEL assay (29) was used for immunohistochemical detection of apoptosis using an ApoTag Peroxidase Kit or an ApoTag Fluorescein Kit from Intergen (Purchase, NY). The TUNEL assay was performed on hepatocytes cultured in 8-well chamber slides, which were fixed in 10% neutral buffered formalin for ~2 h, following which they were transferred to ethanol or processed immediately. Apoptotic nuclei were expressed as the percentage of total nuclei counted, which was 100–150 nuclei/chamber. The liver slices were fixed overnight in 10% neutral buffered formalin. After standard processing for the preparation of sections from paraffin-embedded tissues, the apoptotic hepatocyte nuclei were detected using the TUNEL assay. For slices, each experiment included 3 or 4 slices/treatment. For each slice, a total of 100–200 nuclei were counted.

Detection of high molecular weight (HMW) DNA fragmentation
Field inversion gel electrophoresis was used to detect HMW DNA fragmentation, as described previously (30). Briefly, cultured, precision cut liver slices and hepatocytes treated for 24 h as indicated were harvested and washed three times with ice-cold phosphate-buffered saline (PBS). Slices were individually homogenized on ice in 40 µl cold PBS with 20 strokes of a 1 ml Dounce homogenizer with a tight pestle. Homogenates were mixed with 40 µl of 1.8% pre-melted low melting point agarose (in PBS). Cultured hepatocytes were scraped from the culture dishes and centrifuged. Approximately 2x106 cells were resuspended in 40 µl of PBS, mixed gently with 40 µl of 1.8% pre-melted low melting point agarose and transferred to a plug mold. After refrigeration for 10 min, the plugs were removed from the mold and placed in 5 ml of a solution containing 0.5 M EDTA (pH 8.0), 1% sodium N-lauroylsarcosine plus proteinase K (5 µg/ml) and incubated in a 50°C water bath for 24–30 h. The plugs were then washed twice with TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA) for 1 h each at room temperature and then incubated in 5 ml of TE containing RNase A (5 µg/ml) for 1 h. After briefly washing with TE buffer twice, the plugs were loaded into the wells (1 plug/well) of a 0.8% agarose gel (in PBS) that was pre-chilled thoroughly before use. The gel was run in 0.5x TBE buffer (1x TBE buffer = 45 mM Tris, 45 mM boric acid, 1 mM EDTA) containing ethidium bromide (0.5 µg/ml) with recirculation using program 6 of a PPI-200 field inverter (MJ Research, Watertown, MA) at 150 V. After running for 15 h, the gel was photographed over UV light illumination.

Detection of cytotoxicity
Three standard assays for toxicity, Neutral red uptake (In Vitro Toxicology Assay Kit–Neutral Red; Sigma), reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Cell Titer 96TM Aqueous Non-Radioactive Cell Proliferation Assay Kit; Promega Corp., Madison, WI) and uptake of the impermeant dye propidium iodide (PI) (Molecular Probes, Eugene, OR), were also conducted. Hepatocytes were cultured in triplicate for 24 h in the presence of EE (1, 3 or 10 µM), TGF-ß (3, 5 or 10 ng/ml) and EE (1 µM) + TGF-ß (5 ng/ml) prior to assay.

Detection of cytochrome c release by western blot
Isolation of the 100 000 g cytosolic proteins and detection of cytochrome c release into the cytosol fraction by western blot were performed according to procedures described previously (31). Briefly, cultured rat hepatocytes were harvested by scraping in the culture medium and centrifuged at 750 g for 10 min at 4°C to pellet the previously attached and unattached cells. After resuspension in cold PBS and recentrifugation for 5 min at 4°C, the cell pellets were resuspended in 300 µl of ice-cold buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA) containing freshly added sucrose (250 mM), phenylmethylsulfonyl fluoride (1mM), leupeptin (1mM) and aprotinin (1mM). Cells were homogenized with 50 strokes of a 1ml Dounce homogenizer using the tight pestle. Homogenates were centrifuged for 10min at 4°C at 750 g in a microcentrifuge. Supernatants were transferred to a fresh tube and centrifuged at 10 000 g for 15 min at 4°C. The resulting supernatants were transferred to an ultracentrifuge tube and centrifuged at 100 000 g for 1 h at 4°C. The 100 000 g supernatant was transferred to 1.5 ml microfuge tubes for storage at –80°C. Protein contents of the supernatants were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA) with bovine serum albumin as protein standard.

Approximately 15 µg of 100 000 g supernatant protein was mixed with 10 µl of 2x SDS gel loading buffer (100 mM Tris, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue dye) and boiled for 3 min. The proteins were electrophoretically separated on a 7% SDS–PAGE gel using a mini-gel apparatus (Bio-Rad) at 100 V for 1 h. Gels were electroblotted to nitrocellulose membranes at 25 V overnight. Membranes were first probed with anti-cytochrome c antibody (PharMingen, San Diego, CA) and then with alkaline phosphatase-conjugated secondary antibody (Bio-Rad). The cytochrome c bands were visualized by color development with AP color reagents A and B (Bio-Rad).

Statistical analyses
The data are expressed as means ± SD (n = 3–4) and were analyzed using a one-way ANOVA. Differences were considered significant at P < 0.05 as determined using the Student–Newman–Keuls method for pair-wise multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effects of EE on TGF-ß-induced apoptosis in cultured liver slices
Precision cut liver slices were cultured for 24 h in the presence of 3, 10 or 30 µM EE, 5 ng/ml TGF-ß or EE + TGF-ß (Figure 1AGo). The slices were harvested and processed for determination of percent hepatocytes undergoing apoptosis using the ApoTag Peroxidase TUNEL assay. The background level of apoptosis at time 0, ~5%, did not change significantly over the 24 h culture period. EE caused a concentration-dependent effect on apoptosis. In this experiment, at 3 µM EE the percent TUNEL-positive hepatocyte nuclei was slightly lower than in untreated controls (P < 0.05). In contrast, apoptosis was increased to ~15 and 30% at EE concentrations of 10 and 30 µM, respectively. The percent TUNEL-positive hepatocyte nuclei in the TGF-ß-treated slices was ~20%. In slices exposed to both EE and TGF-ß, 3 µM EE completely prevented induction of apoptosis by TGF-ß. At higher EE concentrations, the percent TUNEL-positive hepatocyte nuclei detected in the cultures exposed to EE plus TGF-ß was similar to that seen with EE alone.



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  		Fig. 1. The effect of EE and TGF-ß on apoptosis in cultured, precision cut rat liver slices. Rat liver slices were prepared and cultured as described in Materials and methods. EE dissolved in ethanol and TGF-ß (10 ng/ml stock in 36% acetonitrile, 0.1% trifluoroacetic acid) diluted in medium were added to the cultures at the concentrations indicated. Controls received only ethanol. After 24 h (A) or 24 and 48 h (B) treatment the slices were harvested, fixed in 10% buffered formalin and processed for use in the TUNEL assay as described in Materials and methods. (A) and (B) represent data from two separate experiments. For each slice 100–200 nuclei were counted to determine the percent TUNEL-positive hepatocyte nuclei present. Each column represents the mean ± SD of three slices. Where no bar is visible, the SD is too small to see. aSignificantly less than untreated control, P < 0.05; bsignificantly less than TGF-ß alone, P < 0.05.

 
Another experiment was conducted to both confirm the low concentration protective effect of EE and to extend the period of observation to 48 h (Figure 1BGo). In controls the percent apoptotic hepatocytes (percent TUNEL-positive hepatocyte nuclei) in cultured slices was approximately 2.5 after 24 h and had not changed significantly at 48 h culture. In this experiment, EE alone at 1 or 3 µM had no significant effects on basal apoptosis at either 24 or 48 h. TGF-ß caused an ~10-fold increase in percent apoptotic nuclei. This TGF-ß effect was completely blocked by simultaneous treatment with EE at 1 and 3 µM at both 24 and 48 h. Similar results were observed in another experiment using the ApoTag Fluorescein TUNEL assay (data not shown).

The results of the TUNEL assays indicated that EE blocked TGF-ß-induced apoptosis in hepatocytes in cultured, precision cut liver slices. Since the TUNEL assay can give false positive results (32), we used another end-point assay for apoptosis to confirm induction by TGF-ß and inhibition by EE in the cultured liver slices. A characteristic of apoptosis is activation of nucleases that cleave DNA initially into domain size HMW DNA fragments. Figure 2AGo shows a photograph of a representative HMW DNA fragmentation gel from cultured, precision cut liver slices treated for 24 h with TGF-ß ± EE. At time 0 in fresh, non-cultured slices an ~48 kb DNA band is clearly visible. A distinct HMW DNA fragment is not visible although there is some smearing, possibly due to the presence of some damaged cells caused by slicing of the tissue. After 24 h a HMW DNA fragment was detected at similar levels in controls and in cultures treated with EE (1 µM). TGF-ß increased the amount of DNA present in the HMW DNA. This TGF-ß effect was inhibited by the simultaneous presence of EE. A negative of this photograph was scanned and percent total DNA present in the fragmented band was determined (Figure 2BGo). These data show that simultaneous treatment with EE significantly protected hepatocytes in the cultured liver slices from TGF-ß-induced HMW DNA fragmentation (apoptosis).



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  		Fig. 2. The effect of EE and TGF-ß on HMW DNA fragmentation in cultured, precision cut rat liver slices. Rat liver slices were prepared and cultured as described in Materials and methods. EE dissolved in ethanol and TGF-ß (10 ng/ml stock in 36% acetonitrile, 0.1% trifluoroacetic acid) diluted in medium were added to the cultures at the concentrations indicated. Controls were treated with the vehicles in which the agents were dissolved. After 24 h the slices were harvested and processed for determination of the extent of HMW DNA fragmentation as described in Materials and methods. (A) A photograph of an ethidium bromide stained gel following electrophoresis. Each lane of the gel represents DNA from a separate liver slice. (B) Percent DNA present in the band representing the HMW DNA fragment as determined by densitometric scans of each lane in a negative of this photograph. The percent of DNA in the HMW DNA fragment band represents the percent of the total amount of DNA present, i.e. that remaining at the origin plus that in the 48 kb band plus that in the HMW fragment band. Each column represents the mean ± SD from three individual slices. Where no bar is visible, the SD is too small to see. aSignificantly greater than the 24 h control, P < 0.05; bsignificantly less than TGF-ß alone, P < 0.05.

 
Effects of EE on TGF-ß-induced apoptosis in cultured rat hepatocytes
The precision cut liver slice culture system presents some limitations to the conduct of the type of mechanistic studies we envisioned. Thus, we investigated the effects of EE and TGF-ß on apoptosis in cultured hepatocytes. Fluorescence microscopy was used to visualize apoptotic nuclei detected by Hoechst dye staining. After an initial 18 h culture period, the hepatocytes were treated with TGF-ß ± EE (1 µM) and harvested 24 h later. Representative photomicrographs of Hoechst dye staining are shown in Figure 3Go. Under these culture conditions the hepatocytes attach and form tight colonies that maintain differentiated functions, including formation of tight junctions and bile canaliculi, as reported previously (33). Normal hepatocyte nuclei are evident and arrows point to apoptotic nuclei. In these representative fields there were fewer apoptotic nuclei in hepatocytes treated with EE + TGF-ß compared with TGF-ß alone. The ApoTag Fluorescein TUNEL assay was then used to quantify apoptosis in the cultured hepatocytes. Hepatocytes were treated with TGF-ß ± EE (1µM) for 24h (Table IGo). In this experiment TGF-ß increased percent TUNEL-positive nuclei 2- to 3-fold above that seen in control and EE-treated hepatocytes. EE completely inhibited the increase in apoptosis caused by TGF-ß. The actual number of TUNEL-positive hepatocytes varied somewhat among different experiments with hepatocytes from individual rats and the level of apoptosis induced by TGF-ß was always less than that seen in liver slices. Nevertheless, the results were consistent in showing that EE suppresses TGF-ß-induced apoptosis. In a separate experiment, the effects of EE (1, 3 and 10 µM), TGF-ß (5 ng/ml) and EE (1 µM) + TGF-ß on cytotoxicity were determined as described in Materials and methods. None of the treatments caused decreases in MTS reduction or Neutral red uptake (data not shown). PI uptake was only increased significantly in hepatocytes exposed to 10 µM EE (data not shown). These results demonstrate that the concentration of TGF-ß used to induce apoptosis (5 ng/ml) and the concentrations of EE that inhibit apoptosis (1 and 3 µM) do not cause toxicity as determined using these three end-points.



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  		Fig. 3. The effect of EE and TGF-ß on apoptosis in cultured rat hepatocytes. Rat hepatocytes were prepared and inoculated into culture as described in Materials and methods. After culture overnight, the medium was changed and EE dissolved in ethanol and TGF-ß (10 ng/ml stock in 36% acetonitrile, 0.1% trifluroacetic acid) diluted in medium were added to the cultures at the concentrations indicated. Controls received only ethanol. Twenty-four hours later the cells were harvested, fixed in 10% buffered formalin and stained with Hoechst dye 33258. The arrows indicate the apoptotic nuclei. Magnification x400.

 

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  		Table I. Effect of EE on TGF-ß-induced apoptosis in cultured hepatocytes detected using the TUNEL assay
 
The effects of EE on TGF-ß-induced apoptosis using two additional end-points, HMW DNA fragmentation and cytosolic cytrochrome c levels, were also determined. Figure 4AGo shows a photograph of a representative HMW DNA fragmentation gel from rat hepatocytes treated for 24 h with TGF-ß ± EE. In controls, only the single 48 kb DNA band was detected and treatment with EE (1 µM) had no effect. TGF-ß caused the appearance of a band representing HMW DNA fragmentation and this effect was completely inhibited by the simultaneous presence of EE. The negative of this photograph was scanned and percent total DNA present in the fragmented band was determined (Figure 4BGo). Inspection of the gel photograph and the data derived from the scans indicate that the level of fragmentation induced in the hepatocytes by TGF-ß was less than that seen in the slices (compare Figures 2 and 4GoGo). This correlates with the reduced apoptotic response detected using the TUNEL assay in hepatocytes versus the slices (compare Figure 1Go and Table IGo). These data show that, similar to what was observed in the cultured liver slices, simultaneous treatment with EE significantly protected cultured hepatocytes from TGF-ß-induced HMW DNA fragmentation (apoptosis).



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  		Fig. 4. The effect of EE and TGF-ß on HMW DNA fragmentation in cultured hepatocytes. Rat hepatocytes were prepared and inoculated into culture as described in Materials and methods. After culture overnight, the medium was changed and EE dissolved in ethanol and TGF-ß (10 ng/ml stock in 36% acetonitrile, 0.1% trifluroacetic acid) diluted in medium were added to the cultures at the concentrations indicated. Controls were treated with the vehicles in which the agents were dissolved. The cells were harvested and processed for determination of the extent of HMW DNA fragmentation as described in Materials and methods. (A) A photograph of the ethidium bromide stained gel following electrophoresis. Each lane of the gel represents DNA from a separate hepatocyte culture. (B) Percent DNA present in the band representing the HMW DNA fragment as determined by densitometric scans of each lane in a negative of this photograph. The percent of DNA in the HMW DNA fragment band represents the percent of the total amount of DNA present, i.e. that remaining at the origin plus that in the 48 kb band plus that in the HMW fragment band. Each column represents the mean ± SD from three individual hepatocyte cultures. Where no bar is visible, the SD is too small to see. aSignificantly greater than the 24 h control, P < 0.05; bsignificantly less than TGF-ß alone, P < 0.05.

 
Release of mitochondrial cytochrome c to the cytosol, where it functions in the activation of caspase 3, is involved in the onset of apoptosis (31). To provide additional evidence in support of the activation of apoptosis by TGF-ß and its inhibition by EE, we used western blot analysis to determine the levels of cytosolic cytochrome c in cultured hepatocytes treated for 24 h with TGF-ß ± EE. Figure 5Go shows a photograph of a western blot representative of three separate experiments. The lower band is 15 kDa cytochrome c and the upper `non-specific' band is a protein reproducibly detected by the secondary antibody. In a separate experiment, it was determined that the intensities of both bands were linear functions of the amount of protein loaded on the gels (data not shown). The intensity of the upper band did not change as a function of treatment and, thus, was used to correct for small differences in signal intensity among lanes. The averages of the duplicate relative intensities of the cytochrome c bands, determined from scanning a photographic negative of this western blot and expressed as a percentage of the controls, are also shown in Figure 5Go. These data show that EE (lanes 3 and 4) alone had no effect on cytosolic cytochrome c levels compared with the controls (lanes 1 and 2). TGF-ß alone increased the level of cytosolic cytochrome c ~2.5-fold (lanes 5 and 6), while in the presence of EE the TGF-ß induction of cytochrome c release was completely inhibited (lanes 7 and 8). These results provide additional evidence that the responses under investigation represent the induction of apoptosis by TGF-ß and its inhibition by EE.



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  		Fig. 5. The effect of EE and TGF-ß on the level of cytosolic cytochrome c in cultured hepatocytes. Rat hepatocytes were prepared and inoculated into culture as described in Materials and methods. After culture overnight, the medium was changed and EE dissolved in ethanol and TGF-ß (10 ng/ml stock in 36% acetonitrile, 0.1% trifluroacetic acid) diluted in medium were added to the cultures at the concentrations indicated. Controls received only ethanol. Twenty-four hours later the cells were harvested and processed for western analysis of cytosolic cytochrome c levels as described in Materials and methods. The figure shows a photograph of the western blot. The relative cytochrome c band densities at the bottom represent scans of a negative of the photograph normalized for the upper `non-specific' band as described in Materials and methods. Each value is the mean of the two lanes for each treatment and the relative band intensity of the controls was set at 1.0. Each lane represents the cytosolic cytochrome c level in a separate hepatocyte culture.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
EE is a strong promoter of hepatocarcinogenesis and, with chronic exposure in some rat strains, is a weak complete hepatocarcinogen (2,34). Our long-term goal is to describe the mechanisms by which EE causes these effects. In previous papers we reported that an initial response to EE treatment is a transient increase in hepatocyte DNA synthesis, through an estrogen receptor-mediated process (3), followed by growth suppression (mitosuppression) (6). TGF-ß and mannose 6-phosphate/insulin-like growth factor II, which facilitates the proteolytic activation of latent TGF-ß (35), were among the genes whose expression was increased during mitosuppression (13,36).

TGF-ß has two major effects on hepatocytes; growth inhibition and induction of apoptosis. In this paper we present data showing that EE inhibits TGF-ß-induced apoptosis in hepatocytes within cultured, precision cut liver slices and in primary monolayer cultures. At low concentrations, i.e. 1 and 3 µM, EE alone had little or no effect on hepatocyte apoptosis whereas TGF-ß-induced apoptosis was inhibited. These results were observed using several end-points for apoptosis, including the TUNEL assay, fluorescent staining with Hoechst dye to visualize apoptotic nuclei, detection of HMW DNA fragmentation and increased levels of cytosolic cytochrome c, providing confidence that it is effects on apoptosis that are being observed. The cultured, precision cut liver slices seemed to be more sensitive to TGF-ß-induced apoptosis since both percent TUNEL-positive hepatocyte nuclei and extent of HMW DNA fragmentation were greater than in the cultured hepatocytes. The reasons for this difference are unknown. However, these are very different types of cultures under different culture conditions. The slices retain the normal architecture and cellular composition of the intact liver. They are cultured in 95% O2, 5% CO2 to curtail necrosis due to hypoxia, although their viability is limited to 48–72 h. The hepatocyte cultures are comprised primarily of hepatocytes and the conditions used for their culture maintain viability and differentiated function for 1–2 weeks. Nevertheless, the same protective effects of EE were observed in both systems, showing that the results are not limited to one culture system. At high concentrations, i.e. 10 and 30 µM, EE alone induced apoptosis to levels even greater than observed with 5 ng/ml TGF-ß. The mechanism of this effect is not known. Assays for Neutral red uptake and MTS activity detected no effects of treatment, whereas uptake of the impermeant dye PI was increased at 10 µM EE, suggesting the possibility of some degree of cytotoxicity. In vivo the effects of EE show a biphasic dose–response effect. At low doses (<=5 µg/day) mitosuppression follows the initial transient increase in hepatocyte proliferation, whereas doses 10–20 times higher are associated with a renewed increase in proliferation (4,5), which may represent a cytotoxic response and enhanced apoptosis.

Exposure of rats to other hepatic tumor promoters, including PB and several peroxisomal proliferators, causes transient growth stimulation followed by mitosuppression (7–11). Thus, while this pattern, i.e. growth stimulation followed by growth inhibition, may not be common to all hepatic tumor promoters (37), the presence of a period when normal hepatocytes are growth inhibited is a common characteristic of hepatic promoters and not unique to EE. An underlying mechanism of mitosuppression appears to involve increased expression of TGF-ß (9,12–14). It is likely, although not proven, that increased expression of TGF-ß and/or its receptors caused by these hepatic promoters causes mitosuppression in normal hepatocytes and that initiated hepatocytes undergoing clonal expansion are characterized by decreased sensitivity to growth inhibition by TGF-ß, as proposed by Jirtle et al. (12) for promotion by PB.

However, TGF-ß also induces hepatocyte apoptosis. Thus, increased TGF-ß during mitosuppression would be expected to cause widespread apoptosis in hepatocytes. This has not been observed, suggesting that apoptosis is being inhibited. Furthermore, during carcinogenesis a function of apoptosis is to eliminate cells that experience excessive damage. Several peroxisome proliferators and EE cause increased oxidative DNA damage (34,38–40) and PB and the peroxisome proliferator ciprofibrate activate hepatic NF-{kappa}B (41,42), a transcription factor known to be activated by oxidative stress (43). Thus, unless apoptosis were inhibited, one might expect that hepatocytes within altered hepatic foci undergoing clonal expansion induced by these promoters would accumulate oxidative damage and then be eliminated by apoptosis. However, PB, several peroxisome proliferators (23–25,44) and, as shown in this study, EE inhibit TGF-ß-induced apoptosis.

The finding that EE inhibits apoptosis in hepatocytes is not surprising since estrogen has been shown to have this effect in other cell types. Estradiol (E2) has been implicated in suppression of spontaneous apoptosis in adult brain neurons (45) and in protection of cultured neural cells expressing mutant presenilins-1 and against apoptosis induced by nerve growth factor withdrawal and exposure to amyloid ß-peptide (46). In the MCF-7 human breast cancer epithelial cell line pretreatment with E2 inhibited apoptosis induced by subsequent exposure to tamoxifen (47). This effect was associated with a 5-fold increase in Bcl-2, an endogenous inhibitor of apoptosis (31). Of interest, Huang and Chou reported that overexpression of Bcl-2 blocked induction of apoptosis by TGF-ß in two human hepatoma cell lines (48). In mouse hepatocytes and liver, Christensen et al. have shown altered expression of members of the Bcl-2 gene family toward expression of those favoring apoptosis following treatment with TGF-ß and toward those favoring inhibition of apoptosis following treatment with PB or nafenopin (24,49). These observations suggest that the role of Bcl-2 induction by EE in inhibition of TGF-ß-induced apoptosis in hepatocytes should be investigated.

In conclusion, during the process of liver tumor promotion by PB, some peroxisome proliferators and EE a transient increase in growth is followed by mitosuppression, which is associated with increased levels of TGF-ß. This suggests that in an environment where TGF-ß levels are increased, growth of normal hepatocytes in response to the promoter is inhibited. In contrast, the ability of initiated hepatocytes to undergo tumor promoter-enhanced clonal outgrowth implies that they are resistant to TGF-ß growth inhibition. This differential response forms the basis of the process of tumor promotion, as suggested by Jirtle et al. (12). At the same time, tumor promoter-mediated inhibition of apoptosis would facilitate continued growth of initiated hepatocytes and, perhaps, in the face of accumulating oxidative damage, their progression. Of interest and possibly of considerable importance, the growth inhibitory and apoptosis-inducing activities of TGF-ß can be separated (50). This provides support for the hypothesis that initiated cells can be insensitive to TGF-ß-induced growth inhibition while remaining sensitive to inhibition of TGF-ß-induced apoptosis caused by the tumor promoter.


   Notes
 
1 Present address: FDA Center for Drug Evaluation and Research, Office of Pharmaceutical Science, Rockville, MD 20855, USA Back

2 To whom correspondence should be addressed Email: jyager{at}jhsph.edu Back


   Acknowledgments
 
This research was supported by US PHS National Institutes of Health Grants R01 CA 36701, P30 ES 03819 and T32 ES 07141, which supported J.C. when he was a post-doctoral fellow.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 24, 1999; revised January 21, 2000; accepted January 31, 2000.


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