Transforming growth factor beta1-induced cell death in preneoplastic foci of rat liver and sensitization by the antiestrogen tamoxifen

Transforming Growth Factor b1 –Induced Cell Death in Preneoplastic Foci of Rat Liver and Sensitization by the Antiestrogen Tamoxifen LEONHARD MU¨LLAUER, BETTINA GRASL-KRAUPP, WILFRIED BURSCH,

Previous studies have shown 5- to 10-fold higher rates of apoptosis in prestages of liver cancer (putative preneoplastic cell foci [PPF]) than in unaltered liver; fasting or withdrawal of tumor promoters enhanced apoptosis even further. We studied whether transforming growth factor b1 (TGF-b1 ), an inducer of apoptosis in normal liver, might be involved in induction of apoptosis in PPF. PPF were produced in 7-week-old female Sprague-Dawley rats with a single oral dose of the genotoxic carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). At 24 weeks of age, TGF-b1 was injected into animals (40 mg/kg intravenously) either once and they were killed 4 hours later (single-dose experiment) or eight times at 24-hour intervals and they were killed 24 hours after the last administration (multiple-dose experiment). Further subgroups received daily subcutaneous injections of tamoxifen (TAM) (8 mg/kg) for 4 consecutive weeks before TGFb1 treatment. In normal liver, the apoptosis incidence was low in solvent- and TAM-only–treated animals, in the single- as well as the multiple-dose experiment. TGFb1 increased the apoptosis incidence severalfold, and the combined administration of TGF-b1 with TAM caused a further strong increase. The already-elevated basal apoptotic incidence in PPF was further increased by TGF-b1 and particularly by TGF-b1 plus TAM treatments, which resulted in a reduction of foci number and size. In summary, these results show that TGF-b1 can induce apoptosis in PPF. This apoptosis-inducing activity is strongly enhanced by the additional treatment with the antiestrogen TAM, which by itself does not have any cell death–inducing effect in the liver or PPF. The elevated apoptotic activity of PPF in response to TGF-b1 can lead

Abbreviations: PPF, putative preneoplastic foci; TGF-b1 , transforming growth factor b1 ; DMBA, 7,12-dimethylbenz(a)anthracene; TAM, tamoxifen; ABs, apoptotic bodies; GST-P, placental glutathione-S-transferase isoenzyme; LI, labeling indices. From the Institute for Tumor Biology–Cancer Research, University of Vienna, Vienna, Austria. Received August 14, 1995; accepted November 1, 1995. Supported by the Austrian ‘‘Fonds zur Fo¨rderung der wissenschaftlichen Forschung.’’ Dr. Mu¨llauer’s present address is: Institute of Clinical Pathology, University of Vienna, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. Address reprint requests to: Wilfried Bursch, Institute of Tumor Biology– Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2304-0027$3.00/0

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to a selective reduction of the liver load with preneoplastic cells. (HEPATOLOGY 1996;23:840-847.)

Foci of phenotypically altered hepatocytes are widely considered to represent prestages of liver cancer in humans and experimental animals.1-5 In rat liver, these putative preneoplastic foci (PPF) have been well characterized and provide a useful model to study compound effects on carcinogenesis. PPF show enhanced rates of cell replication and apoptosis6-11; treatment with tumor promoters enhances replication and inhibits apoptosis, resulting in preferential foci growth during tumor promotion.2,10 Conversely, promoter withdrawal or food restriction decrease replication and enhance apoptosis in foci2,8,12,13; this may lead to a reduction of foci volume and complete extinction of foci. Little is known about growth regulation in foci by endogenous factors. Recently, it has been found that transforming growth factor b1 (TGF-b1 ), known as a stimulator of mesenchymal and inhibitor of epithelial cell proliferation,14,15 can induce apoptotic cell death. In normal rat liver, TGF-b1 appears in cells preparing for apoptosis,16,17 and it triggers apoptosis of unaltered hepatocytes in vitro and in vivo.18,19 In the present study, we asked whether TGF-b1 can also induce apoptosis in PPF. Female Sprague-Dawley rats were used as the model. They were treated with a single oral dose of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), which leads to the development of PPF and breast carcinomas.20 In previous studies, induction of apoptosis by TGFb1 was particularly effective during involution of liver hyperplasia following treatment with a hepatomitogen such as cyproterone acetate.18,19 Apparently, induction of apoptosis is favored after mitogen withdrawal. Consequently, we sought to determine whether treatment with antihormones, which antagonize endogenous mitogens, would likewise commit hepatocytes and breast cancer cells to apoptosis and make them more susceptible toward TGF-b1 . Estrogens are mitogenic to liver21,22 and breast tissue23; therefore, we have used the antiestrogen tamoxifen (TAM) to antagonize endogenous estrogens. In this report, we describe our observations of TGF-b1’s effects on the liver and focus on our results regarding the following questions: (1) Are PPF sensitive toward growth inhibition and/or apoptosis induc-

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tion by TGF-b1 ? (2) Does an antiestrogenic substance such as tamoxifen modify TGF-b1 effects on an estrogen-responsive organ such as the liver? (3) Are putative preneoplastic cells different in their sensitivity toward TGF-b1 as compared with normal hepatocytes? and (4) Can TGF-b1 effectively lower the liver’s burden with preneoplastic cells? The results obtained with breast cancer tissue will be reported elsewhere.2 MATERIALS AND METHODS Experimental Design. Three-week-old, pathogen-free, female Sprague-Dawley rats were obtained from the ‘‘Forschungsinstitut fu¨r Versuchstierzucht und -haltung’’ of the University of Vienna (Himberg, Austria). Animals were kept under standardized conditions (Macrolon cages, 12hour–light phase with the light off from 10 AM to 10 PM, 23 { 27C room temperature, 40 to 70% relative humidity), and received a standard diet (Altromin, Lage, Germany) and water ad libitum. A single dose of 130 mg/kg body weight of DMBA (Sigma Chemie, Deisenhofen, Germany), dissolved in 10 mL maize oil/kg, was administered by a gastric tube to 49- to 51-day-old rats. For a 6-hour period prior to DMBA administration, rats had access only to drinking water. TAM treatment was started 13 weeks after DMBA administration and 4 weeks before the start of TGF-b1 injections, and was continued throughout the TGF-b1 treatment period. TAM (ICI Pharmaceuticals, Macclesfield, England) was dissolved in 20% wt/wt benzylbenzoate and 80% wt/wt castor oil. Eight mg TAM/kg b.w. in 1 mL were injected daily and subcutaneously into the animals’ backs. Control animals received TAM and TGF-b1 solvents (0 / 0) or TAM and TGF-b1 solvent (TAM / 0). Animals of the two other experimental groups received TAM solvent and TGF-b1 (0 / TGF-b1 ) or TAM and TGF-b1 (TAM / TGF-b1 ). DNA synthesis in the liver is synchronized to a single peak per day through rhythmic feeding.24 Therefore, a feeding rhythm was introduced 2 weeks before the first TGF-b1 administration with food available from 10 AM to 7 PM. Recombinant mature TGF-b1 was obtained from Bristol-Myers-Squibb (Seattle, WA). TGF-b1 was dissolved in 5 mmol HCl in 10 mmol Tris/HCl (pH 7.4), containing 1 mg of bovine serum albumin (BSA) per milliliter. As control, 5 mmol HCl in 10 mmol Tris/HCl containing 1 mg of BSA per mL was injected. TGF-b1 was injected into a tail vein at a dose of 40 mg/mL/kg of body weight either for 8 consecutive days at 10 AM, with the animals killed at 10 AM on the ninth day (multiple-dose experiment), or once at 6 AM, with the animals killed 4 hours later (single-dose experiment). In the long-term experiment, DNA synthesis activity was monitored by measuring the [3H]thymidine incorporation into nuclear DNA. [3H]Thymidine (6.7 Ci/mmol/L) was purchased from NEN (Vienna, Austria), diluted in 0.9% NaCl, and injected intraperitoneally at a dose of 0.2 mCi/kg. Injection times were 1 PM, 9 PM, and 5 AM the next day when the animals were anesthetized in CO2 and decapitated at 10 AM. The number of animals per experimental group had to be limited to three in the multiple-dose experiment and four or five (TAM / TGF-b1 ) in the single-dose experiment due to limited supply of TGF-b1 . The animal experiments were performed according to the Austrian guidelines for animal care and treatment. Liver DNA and Protein Content. At necropsy liver weights were recorded; 0.5-g aliquots were snap-frozen in liquid nitrogen for the biochemical determination of hepatic DNA and protein content as described previously.24 Histology. Tissues were fixed in Carnoy’s fixative, embed-

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ded in paraplast, and 2- to 4-mm serial sections were cut. One section was stained with hematoxylin-eosin and served to determine the frequency of apoptotic bodies (ABs). A second section was used for the detection of placental glutathioneS-transferase isoenzyme (GST-P) expression to identify PPF and to measure their number and size.25,26 A third section was used to determine labeling indices (LI) as described previously.27 The identification of ABs followed previous descriptions.28 The incidence of ABs in unaltered liver tissue was evaluated by scoring at least 2,000 periportal and perivenous hepatocytes and expressed per 100 intact hepatocytes (% ABs). PPF were identified as groups of hepatocytes expressing placental glutathione-S-transferase isoenzyme (GST-P) and in hematoxylin-eosin–stained sections according to published criteria.25,26 DNA synthesis in the liver was determined by counting the number of labeled hepatocytes per 100 nucleated hepatocytes (the LI). LI were assayed on at least 2,000 unaltered hepatocytes in each liver and in all nucleated cells of individual foci. Serum Activities of Liver Enzymes. Serum of each animal was prepared by centrifugation (15 minutes, 3,000 rpm, 47C) and stored at 0707C. The activities of aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and gamma-glutamyl transferase were determined by standard procedures at the First Clinic of Internal Medicine of the University of Veterinarian Medicine, Vienna, Austria. Statistical Analysis. Means and SD of control and treated groups were calculated. The comparison between corresponding groups was performed by the two-tailed Student’s t test. Means and 95% CI are given for apoptosis and LI. RESULTS Effects of TGF-b1 on Body Weight and Liver Mass.

Repeated doses of TGF-b1 caused a marked bodyweight loss, particularly in TAM-treated rats (0 / TGFb1 , TAM / TGF-b1 ), whereas the body weights of solvent (0 / 0)- and TAM-only–treated animals (TAM / 0) remained unaltered (Fig. 1). The mean relative liver weights of 0 / TGF-b1- and TAM / TGF-b1 –receiving animals were less than that of the respective control animals (Fig. 2). This reduction of liver weight was paralleled by a decrease in the livers’ DNA and protein content (Fig. 2). This indicates that an actual loss of liver cells had occured. Effect of TGF-b1 on Cell Proliferation and Death in the Liver. In unaltered liver the incidence of ABs was

low in 0 / 0–treated animals in the single- and multiple-dose experiment (Fig. 3A and 3B). A slightly lower incidence of ABs in TAM / 0–receiving animals in the single-dose experiment (Fig. 3A) did not differ significantly from 0 / 0–treated animals and was not seen in the multiple-dose experiment (Fig. 3B). 0 / TGF-b1 administration increased the incidence of ABs in the single-dose experiment (Fig. 3A) several-fold and, although less pronounced, in the multiple-dose experiment (Fig. 3B). The combination of TGF-b1 with TAM caused a further massive increase of apoptotic cell death in the single- and multiple-dose experiment (Fig. 3A and B). This strong enhancement of the apoptosisstimulating activity of TGF-b1 by TAM is not a simple additive effect, because TAM alone did not induce apoptosis at all. In the multiple-dose experiment, only additional cells with morphological characteristics of

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erably increased in 0 / TGF-b1 animals (Fig. 3B). In PPF the DNA synthesis activity was severalfold higher than in unaltered liver in 0 / 0, TAM / 0, and 0 / TGF-b1 treatment groups, and again highest in 0 / TGF-b1 animals (Fig. 3B). This paradoxical stimula-

FIG. 1. Effect of TGF-b1 and TAM treatments on body weight. Animals pretreated with solvent or TAM for 4 weeks received intravenous injections of TGF-b1 (40 mg/kg/day) for a period of 8 consecutive days and were killed on the ninth day (multiple-dose experiment). The body weight before TGF-b1 treatment was set as 100%. Means { SD are shown; the number of animals per group was three. 0 / 0 Å solvent, 0 / TGF-b1 Å solvent plus TGF-b1 , 0 / TAM Å solvent plus TAM, TAM / TGF-b1 Å TAM plus TGF-b1 treatment.

necrosis were observed in unaltered liver of TAM / TGF-b1 animals (mean incidence, 0.78%; data not shown). We further asked whether the different zones of the liver lobule might have a different sensitivity toward induction of apoptosis. We found a higher incidence of ABs in perivenous than in periportal areas (Table 1; only data of the TAM / TGF-b1 group for which the high apoptosis incidence permitted statistical analysis are shown). A preferential centrilobular incidence of apoptosis also has been observed by Benedetti et al.29 in normal human and rat liver, and by Schwall et al.30 in rats after intravenous injection of activin, a protein with structural homology to TGF-b1 . In PPF the incidence of ABs generally was severalfold higher than in the surrounding unaltered liver for all four treatment groups in the single- as well as the multiple-dose experiment (Fig. 3A and 3B). No difference in the apoptotic activity was found between GST-P–positive and GST-P–negative PPF (data not shown). Furthermore, no signs of necrosis were observed in PPF. We also measured, as an index of cell proliferation, the percentage of hepatocellular nuclei incorporating [3H]thymidine (LI) in the multiple-dose experiment. The LI in unaltered liver were similarly low in the groups 0 / 0, TAM / 0, and TAM / TGF-b1 , but consid-

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FIG. 2. Effects of TGF-b1 for 8 days and TAM on relative liver weight, DNA, and protein content. Means {SD are shown, *P õ .05; experimental groups as defined in legend to Fig 1.

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FIG. 3. Effects of TGF-b1 and TAM on the incidence of ABs (A and B) and DNA synthesis (B) in unaltered liver and PPF. Animals pretreated with solvent or TAM for 4 weeks received a single intravenous injection of TGF-b1 and were killed 4 hours later (single-dose experiment). A total of 8,000 to 15,000 unaltered hepatocytes and 1,816 to 6,028 focus cells were evaluated per group. There were five animals in the TAM / TGF-b1 group and four in the other groups (A). Experimental groups as defined in legend to Fig 1. Twelve thousand unaltered hepatocytes and 4,557 to 6,918 focus cells per group were evaluated for the determination of the incidence of ABs. Six thousand to 12,000 unaltered hepatocytes and 681 to 3,869 focus cells per group were evaluated for the determination of the LI. Means with 95% CI are shown.

tion of liver DNA synthesis by TGF-b1 , which has been shown to be a potent inhibitor of hepatocyte proliferation in vitro31-33 and in vivo,34,35 may reflect a regeneration mechanism of the liver to compensate the TGFb1 –induced cell loss. The absence of an increased DNA synthesis activity in TAM / TGF-b1 –treated animals is most likely due to an inhibition of regenerative hepatocyte proliferation36 and sensitization of hepatocytes to the negative growth effects of TGF-b1 by TAM. Elimination of PPF from the Liver. We determined the number and volume of PPF in the liver of the different treatment groups. Results are shown in Fig. 4. The mean number and volume of foci per liver was reduced to approximately one third in 0 / TGF-b1– and TAM / TGF-b1 –treated animals as compared with 0 / 0

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and TAM / 0 groups. These differences failed to be statistically significant (P Å .062 and P Å .085 for the differences in number and volume of PPF between control [0 / 0; 0 / TAM]- and TGF-b1 [0 / TGF-b1 ; TAM / TGF-b1 ]-treated groups) because of the considerable biological variation in liver foci determination. In TAM / TGF-b1 animals the number and volume of foci tended to be less than in 0 / TGF-b1 animals, although the difference was not as pronounced as might be expected. This could be caused by interindividual variations in the number and size of foci before the start of TGF-b1 and/or to differences in the time course of hepatocyte proliferation during the 9-day observation period. In any case, the results support the conclusion that the higher rate of TGF-b1 –induced apoptosis in

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TABLE 1. Zonal Distribution of Apoptotic Cell Death in TGF-b1 Plus TAM-Treated Animals

Single-dose experiment Multiple-dose experiment

Treatment

Periportal (%)

Perivenous (%)

TAM / TGF-b1

30 { 10.7

69.7 { 10.7

TAM / TGF-b1

21 { 10.3

79 { 10.3

Animals pretreated with TAM for 4 weeks were injected TGF-b1 (40 mg/kg) intravenously either once and killed 4 hours later (singledose experiment) or eight times (40 mg/kg/day) in 24-hour intervals and killed 24 hours after the last application (multiple-dose experiment). Percentages were calculated from 315 (single-dose experiment) and 289 (multiple-dose experiment) apoptotic hepatocytes in nonfocal areas. Means { SD are shown. There were five animals in the single-dose and three in the multiple-dose experiment. Abbreviation: TAM / TGF-b1 Å tamoxifen plus TGF-b1 treatment.

accordance with this assumption the number and volume of PPF were reduced in TGF-b1-treated animals. This reduction of PPF was quantitatively more pronounced than the reduction of the livers’ DNA content, which supports the preferential susceptibility of PPF cells to induction of apoptosis by TGF-b1 . Unexpectedly, DNA synthesis activity was increased in animals receiving multiple doses of TGF-b1 , although numerous experiments firmly established TGF-b1 as an inhibitor of hepatocyte proliferation.31-35 Our result is similar to observations made by Russel et al.34 in rats after partial hepatectomy, in which TGF-b1 inhibited the

PPF as compared with the surrounding unaltered liver led to an effective reduction of PPF even within the relatively short treatment period of 8 days. The relative size distribution of the remaining PPF in the 0 / TGFb1 and TAM / TGF-b1 groups was similar to 0 / 0– and TAM / 0–treated animals (data not shown). Release of Liver Enymes Into the Blood After Application of TGF-b1 . As a biochemical indicator of the ex-

tent and nature of a possible TGF-b1 –induced injury to the liver, we measured the serum activities of aspartate transaminase, alanine transaminase, glutamate dehydrogenase, and gamma-glutamyl transferase. In TGFb1 / 0–treated animals no significant increase in the serum levels of the four enzymes, as compared with 0 / 0– and TAM / 0–receiving animals occurred. Treatment with TAM / TGF-b1 elevated the serum levels of AST and ALT approximately twofold. The glutamate dehydrogenase concentration was only modestly increased and g-glutamyl transferase levels were not altered (Table 2). This small increase of hepatic enzymes is far less than that observed after toxic liver injury, e.g., after CCl4 exposure, where aspartate transaminase serum levels ranged from 1,000 to 2,500 U/L.37 The modest increase of hepatic enzymes in this study was not nearly as steep as the increase in apoptosis incidence and coincided with the observation of necrosis. Therefore, it may be assumed that the small release of intracellular material is predominantly due to necrosis and not to apoptosis. This is further supported by the single-dose experiment in which no necrosis of hepatocytes was observed and serum enzyme concentrations were not elevated (data not shown). DISCUSSION

The present study shows for the first time that cells in PPF are stimulated to undergo apoptosis by TGFb1 . Furthermore, as in previous studies,6-10 basal rates of apoptosis and cell proliferation were several-fold higher in PPF than in unaltered liver. Therefore, an increase of the apoptosis rate should result in a greater loss of cells from PPF than from unaltered liver.12 In

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FIG. 4. Effects of TGF-b1 and TAM on the number and volume of PPF. Experimental groups as described in legend to Fig 1. Means (SD) are shown. P values are .062 and .085 for the differences in number and volume of PPF between control (0 / 0; 0 / TAM) and TGF-b1 (0 / TGF-b1 ; TAM / TGF-b1 )-treated groups (two-tailed Student’s t test).

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HEPATOLOGY Vol. 23, No. 4, 1996 TABLE 2. Effect of TGF-b1 for 8 Days and TAM on Serum Enzymes Enzyme Treatment

AST

0/0 0 / TGFb1 TAM / 0 TAM / TGFb1

127 100 87 203

{ { { {

ALT

57 25 16 39

24 19 22 47

{ { { {

7 1.8 3 12

GLDH

5.8 4.0 3.7 6.9

{ { { {

1.4 0.8 6.9 1.0

g-GT

1.0 1.0 2.0 1.0

{ { { {

0.0 0.0 1.7 0.0

Animals pretreated with solvent or TAM for 4 weeks were with TGF-b1 (40 mg/kg/d) intravenously eight times in 24-hour intervals and killed 24 hours after the last application. Means { SD are shown. The number of animals per group was three. Abbreviations: AST, 0 / 0 Å solvent, 0 / TGFb1 Å solvent plus TGFb1, 0 / TAM Å solvent plus tamoxifen, TAM / TGF-b1 Å tamoxifen plus TGF-b1 treatment.

early proliferative response but failed to continuously suppress hepatocyte proliferation. The failure of TGFb1 to continuously inhibit hepatocyte proliferation may result from a number of causes. TGF-b1 has a plasma half-life of only 2 minutes and is rapidly metabolized by the liver,38,39 which may allow hepatocytes to escape from the inhibitory TGF-b1 effects within the injection intervals. The rapid uptake and metabolism of biologically active TGF-b1 by the liver may also, at least in part, have been responsible for a lack of apoptosis-inducing activity in breast carcinomas.2 Furthermore, the timing of administration may have been inadequate for suppression of DNA synthesis because the hepatocyte loss caused by repeated TGF-b1 injections may have stimulated asynchronous regenerative DNA synthesis resistant to TGF-b1 , which acts primarily in the G1 phase of the cell cycle.40 Additionally, there may be temporal changes in the sensitivity of hepatocytes to growth inhibition by TGF-b1 , as suggested by Carr et al.,32 who observed a reduction in TGF-b1 binding 24 hours after partial hepatectomy. In TGF-b1 plus TAM-treated animals a stimulation of hepatic DNA synthesis did not occur, most likely reflecting a synergistic inhibiting influence of TGF-b1 and TAM on hepatocyte proliferation.36 Another important finding of this study was that the antiestrogen TAM enhanced severalfold the potency of TGF-b1 to induce apoptosis. This was not an additive effect because TAM on its own did not induce apoptosis. Thus, an increase of the hepatocytes’ sensitivity to TGF-b1 through TAM is more likely. Several causative mechanisms may be postulated: 1. The liver is a steroid hormone–responsive organ41; hepatocytes contain estrogen receptors42 and respond with proliferation to estrogenic stimulation.21,22 Furthermore, estrogens and certain contraceptive steroids exert a tumor-promoting action in rodent liver21 and induce, albeit rarely, liver tumors in women.43 TAM competes effectively for estradiol binding to estrogen receptor in human liver,44 inhibits hepatocyte proliferation after partial hepatectomy,36 and suppresses the enlargement of hyperplastic nodules in rats.45

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Thus, TAM may have blocked the action of endogenous mitogenic estrogens and thereby signaled to hepatocytes a state of mitogen deficiency, a condition favoring the induction of apoptosis.8,10,18,28 2. TGF-b1 binds to its type II receptor, and intracellular signalling requires both a membrane translocation of the type II receptor and association with the type I receptor.46 TAM alters membrane fluidity,47 which might facilitate receptor interaction, thus increasing the hepatocyte’s sensitivity toward TGF-b1 . 3. TAM may—as in human breast cancer cell lines48 and in the stroma of human breast carcinomas49 —induce TGF-b1 and/or TGF-b1 receptor expression in the liver, thus transforming the hepatocytes into a state more susceptible to TGF-b1 . This would thereby allow the threshold level necessary for apoptosis induction to be surpassed. 4. DNA damage is a well-established trigger for apoptosis through a process involving the tumor-suppressing gene product p53.50 TAM damages DNA,51 and this may induce expression of effectors of apoptosis, whose increased intracellular availability might facilitate stimulation of apoptosis by TGF-b1 . The increased propensity of PPF for apoptosis resulted in a reduced load of the liver with preneoplastic cells. In previous studies, a reduction of PPF was shown to diminish the long-term risk of cancer.12 It remains to be seen whether a reduction of PPF by TGF-b1 early in the carcinogenesis process would likewise protect from tumor formation. The sensitivity of PPF toward TGF-b1 as observed in our study seems to contradict a report by Stenius,52 which stated that GST-P–positive cells isolated from diethylnitrosamine-treated rats are unresponsive to inhibition of DNA synthesis by TGFb1 . This discrepancy could be because of the differing conditions of in vivo and in vitro systems or other variables. The clinical and experimental evidence implicating estrogens in the regulation of hepatic cell proliferation stimulated clinical trials with TAM as an adjuvant therapeutic for hepatocellular carcinoma. Engstrom et al.53 failed to confirm a therapeutic benefit for an unselected group of patients. On the other hand, both a recent clinical trial in cirrhotic patients with unresectable hepatocellular carcinoma54 and two independent case reports55,56 indicated a prolonged survival of TAMtreated patients. From our data, one could postulate a combination of TAM and TGF-b1 and assume an induction of apoptotic cell death in hepatocellular carcinoma. Future studies should determine whether hepatocellular carcinomas are still sensitive toward TGF-b1 –induced apoptosis. Although several reports clearly establish TGF-b1 as a potent inhibitor of the proliferation of normal hepatocytes, current data on the sensitivity of hepatocellular carcinoma cells toward growth inhibition by TGF-b1 are somewhat conflicting.11 Transformed hepatocytes generated from F344 rats by the Solt-Farber model were growth-inhibited by TGF-b1 in vitro,57 and several hepatoma cell lines are sensitive toward growth inhibition and apoptosis induction by

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TGF-b1 .58,59 Conversely, some subclones of rat liver epithelial cells transformed in vitro by selective growth conditions and oncogene transfer were resistant to TGF-b1 .60 However, the response of cell lines established under long-term, selective culture conditions may have only limited relevance for the biology of hepatocellular carcinomas. Any considerations regarding a potential application of TGF-b1 for liver cancer prevention or therapy must take into account systemic side effects of TGF-b1 ,61 such as body-weight loss, which also was observed in this study, organ fibrosis, and immunosuppression. Future experiments should evaluate whether and to what extent other antiestrogenic substances will achieve a cell-death–enhancing effect. Further, the induction of DNA lesions by administration of high doses of TAM must be addressed; new antiestrogens, such as the TAM derivative toremifene, which lacks DNA-modifying and liver carcinogenic activity in rodents,62 may provide an alternative. In summary, the present results show that PPF can be eliminated through the induction of apoptosis by TGF-b1 and show that the antihormonal substance TAM dramatically enhances the intrinsic susceptibility of preneoplastic cells to undergo TGF-b1 –stimulated cell death. These observations suggest the feasibility of therapeutic concepts aimed at eliminating preneoplastic cells via the induction of apoptosis by the administration of endogenous regulators of cell death. Acknowledgment: The excellent technical assistance of K. Bukowska is gratefully acknowledged. R. Tiefenbacher was of great help in the initial phase of the animal experiments. For the generous supply of TGF-b1 , we thank Bristol-Myers-Squibb, USA. REFERENCES 1. Pitot HC. Altered hepatic foci: their role in murine hepatocarcinogenesis. Annu Rev Pharmacol Toxicol 1990;30:465-500. 2. Schulte-Hermann R. Tumor promotion in the liver. Arch Toxicol 1985;57:147-215. 3. Marsman DS, Popp JA. Biological potential of basophilic hepatocellular foci and hepatic adenoma induced by the peroxisome proliferator Wy-14,643. Carcinogenesis 1994;15:111-117. 4. Deugnier YM, Charalambous P, Le Quillcuc D, Turlin B, Searle J, Brissot P, Pokell LW, et al. Preneoplastic significance of hepatic iron-free foci in genetic hemochromatosis: a study of 185 patients. HEPATOLOGY 1993;8:1363-1369. 5. Okuda K. Hepatocellular carcinoma: recent progress. HEPATOLOGY 1992;15:948-963. 6. Schulte-Hermann R, Timmermann-Trosiener I, Schuppler J. Response of liver foci in rats to hepatic tumor promoters. Toxicol Pathol 1982;10:63-70. 7. Bursch W, Lauer B. Influence of liver tumor promoters on cell death by apoptosis in rat liver. Naunyn-Schmiedebergs Arch Pharmacol 1983;322:R119. 8. Bursch W, Lauer B, Timmermann-Trosiener I, Barthel G, Schuppler J, Schulte-Hermann R. Controlled death (apoptosis) of normal and putative preneoplastic cells in rat liver following withdrawal of tumor promoters. Carcinogenesis 1984;5:453-458. 9. Columbano A, Ledda-Columbano GM, Rao PM, Rajalakshmi S, Sarma DSR. Occurrence of cell death (apoptosis) in preneoplastic and neoplastic liver cells. Am J Pathol 1984;116:441-446. 10. Schulte-Hermann R, Timmermann-Trosiener I, Barthel G, Bursch W. DNA synthesis, apoptosis, and phenotypic expression as determinants of growth of altered foci in rat liver during phenobarbital promotion. Cancer Res 1990;50:5127-5135.

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WBS: Hepatology