Influence of Liver Tumor Promoters on Apoptosis in Rat Hepatocytes Induced by 2-Acetylaminofluorene, Ultraviolet Light, Or Transforming Growth Factor /311

(CANCER RESEARCH 56, 1272-1278. March 15. 1996] Influence of Liver Tumor Promoters on Apoptosis in Rat Hepatocytes Induced by 2-Acetylaminofluorene, Ultraviolet Light, or Transforming Growth Factor /311

Wolfgang Worner and Dieter Schrenk2

InsliliiU' of Toxicology, University of Tubingen, Willieltnsiruxse 56, D-72074 TÃ¼bingen,Germany

ABSTRACT acetate from rats bearing preneoplastic liver lesions resulted in a sharp rise in apoptosis within the foci. This finding argued strongly for an DNA damage is recognized widely as a cause of programmed cell death apoptosis-suppressing action of these agents. (apoptosis), aimed at eliminating cells bearing genotoxic lesions. There The most potent tumor promoter in rodent liver is TCDD' (10-12). fore, inhibition of DNA damage-induced apoptosis may play an important role in carcinogenesis and has been suggested as a mechanism of action of In contrast to most other liver tumor promoters, however, TCDD tumor-promoting agents. In the present study, the effects of treatment showed no or very restricted enhancement of hepatocellular prolifer with UV light or the carcinogenic aromatic amine 2-acetylaminofluorene ation in the normal rat liver (13). Similarly, TCDD failed to enhance (2-AAF) on apoptosis were studied in rat hepatocytes in primary culture. DNA synthesis in normal rat hepatocytes in primary culture, although A significantly increased incidence of apoptotic nuclei, showing condensed it enhanced the proliferative response of the cells to epidermal growth or fragmented chromatin visualized with the fluorescent dye Hoechst factor slightly (14). Recently, Stinchcombe et al. (15) found a marked 33258, was found after each type of treatment. After 48 h, the incidence of suppression of apoptosis in glutathione S-transferase-P-positive pre apoptosis had returned to the control level. When the liver tumor pro neoplastic liver foci of TCDD-treated rats. moters 2,3,7,8-tclrachlorodihcn/.o-/7-dioxin (TCDD) and phÃ©nobarbital A variety of stimuli are reported to induce apoptosis in cell culture were added to the medium, Â¡ipoptosisdid not increase in UV- or 2-AAF- (16, 17). TGF-/31 has been shown by Oberhammer et Â¡il.(18) to treated compared with untreated cultures. Furthermore, Kill) and phÃ© nobarbital suppressed internucleosomal DNA fragmentation elicited by induce apoptosis in cultured rat hepatocytes. Another stimulus used in the present study is treatment with DNA-damaging agents. It was UV irradiation. In contrast, the promoters did not suppress apoptosis induced by transforming growth factor /Â¡I.Immunoprecipitation of the found that TGF-ÃŸl. UV irradiation, and the genotoxic aromatic amine tumor suppressor gene product p53 demonstrated that the increase in p53 2-AAF initiated apoptosis in rat hepatocytes, and that apoptosis elic observed after 1 \ irradiation was abrogated almost completely by TC'DD. ited by UV or 2-AAF treatment could be suppressed by the liver Apoptosis induced in rat hepatocytes by DNA-damaging agents such as tumor promoters TCDD and phÃ©nobarbital. UV light or 2-AAF is suppressed by TC'DD and phÃ©nobarbital. Inhibition of apoptosis allowing survival of hepatocytes hearing genotoxic lesions may be crucial for the tumor-promoting action of TC'DD and phÃ©nobar MATERIALS AND MKTHODS bital in the liver. Materials. Hoechst H33258 and 2-AAF were from Sigma Chemical Co. INTRODUCTION (Taufkirch.cn. Germany); TGF-ÃŸl was from Bcclnn Dickinson (Heidelberg, Germany); and protcinasc K and pancreatic RNAsc were from Boehringer In addition to proliferation, programmed cell death (apoptosis) is Mannheim (Mannheim. Germany). Monoclonal anti-pS3 antibodies were from Dianova (Hamburg, Germany); DMFM and Waymoulh's medium 705/1 were the major factor that contributes to the actual growth of a cell clone (1-3). Clonal growth is a characteristic feature of phenotypically from Seromecl (Berlin. Germany): and PCS was from GIBCO (Fggenslein, altered, preneoplastic hepatocytes in the rodent liver (4). These cells Germany ). Rat Hepatocyte Culture and Treatment. Male Wistar rats were from are thought to carry one or more critical hut still undefined lesions in Savo (Kisslegg, Germany) and were kepi on lap water and a standard diet their genome resulting, e.g., from the action of genotoxic liver car (Altromin, Lage, Germany). Hepatocytes were isolated from animals weighing cinogens. Under the influence of liver tumor promoters, the clonal 150-200 g by sequential perfusion of the liver with HOT A- and collagenase/ expansion of preneoplastic liver foci is facilitated, an effect that is calcium-containing buffers via the portal vein as described (14). Hepatocyte apparently related to a further advance on their way to malignancy (5, preparations showing a trypan blue exclusion rale of >909i were seeded on 6). Because application of most liver tumor promoters to rodents leads 90-nim l'etri dishes in a 1:1 mixture of DMKM:Waymouth's 705/1 medium to an initial increase in liver si/.e and to an enhanced proliferation of containing 10 DIMHF.PF.S, 50 IIMdexamethasone. a mixture of 5 fxg/ml each hepatocytes (7), growth stimulation was regarded as a hallmark of of insulin, transform!, and selenous acid (ITS prcmix: Collaborative Research. Lexington, MA). 10% PCS, and 50 jug/ml gentamicin. Por preparation of liver tumor promotion. However, under conditions of sustained ap DNA, cells were plated on 150-mm dishes (Falcon, Oxnard. C'A) al a density plication, e.g., of phÃ©nobarbital, proliferation of both normal and of 100.000 cells/cm'. After 3 h, the medium was replaced by a 1:1 mixture of preneoplastic hepatocytes returns to the initial level (8). Furthermore, DMKM:Waymouth's 705/1 medium supplemented with K) IHMHHPFS, 50 nw the permanently increased rate of proliferation in preneoplastic foci dcxamelhasone. a mixture of 6.25 /xg/ml each of insulin, transferrin, and would lead theoretically to a much higher rate of expansion than selenous acid. 1.25 mg/ml BSA, and 5.35 ^ig/ml linoleic acid (ITS' premix; observed, and the concept of apoptosis counterbalancing proliferation Collaborative Research), and 50 fig/ml gentamicin. Then, apoplosis was ini was brought into the discussion (8, 9). In fact, preneoplastic foci were tiated by addition of TGF-ÃŸl or 2-AAF (Fig. 1). After 12 h, the medium was shown to exhibit a higher incidence of apoptosis, i.e., an increased replaced by fresh medium without TGF-ÃŸl or 2-AAF, and the rale of apoplolic number of apoptotic bodies (8). Bursch et al. (9) demonstrated that nuclei was determined at various time points using fluorescence microscopy. withdrawal of the promoting agents phÃ©nobarbitaland cyproterone For UV treatment, cultures were irradiated 30 min prior lo Ihc second medium change. After the second medium change, TCDD dissolved in DMSO or phÃ©nobarbitaldissolved in saline was added if indicated. Control cultures were Received IO/4TO; accepted l/lh/96. The costs ill' puhlieuticm of this article were defrayed in part by the payment of page treated with DMSO or saline only. charges. This article must therefore he hereby marked udvertisemenl in accordance with Microscopical Detection of Apoptosis. For morphological determination IH U.S.C. Section 1734 solely lo indicate this fact. of the incidence ot apoptosis, the medium was removed, and colls woro fixed 1This work was supported in part by a grant from the Deutsche Korschungsgemein- schaft (Homi, Germany). -Ju whom requests for reprints should he addressed. Phone: 49-7071-294945; Fax: 1The abbreviations used are: TCDD. 2,3.7.S-lc(rachlorodihcnÂ«i-/>-dioxin; 2-AAF, 49-7071-292273. 2-acetylaminofluorene; TOH-/JI, transforming growth factor 01; J. joule. 1272

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. INHIBITION OF APOPTOSIS BY LIVER TUMOR PROMOTERS and stained as described by Oberhammer et al. (18). Cell layers were inspected RESULTS using a Laborlux 12 microscope (Leitz, Wetzlar. Germany) with a Ploemopack fluorescence unit (B2 filter; excitation wavelength, 350-410 nm). Fluorescent In untreated cultures of rat hepatocytes, and to an increased extent nuclei were screened for normal morphology (unaltered chromatin). and ap- after treatment with apoptogenic agents, various patterns of apoptotic optotic nuclei comprising those with fragmented (scattered) and condensed nuclei were observed. The most common type of apoptotic nucleus chromatin were counted. Apoptosis was expressed as the percentage of apop- was that showing droplike, scattered chromatin (Fig. 2). In fluores totic nuclei/10' nuclei. cence and visible microsocopy, the apoptotic hepatocyte surrounding DNA Fragmentation Analysis. Hepatocyte cultures were treated as shown the nucleus in most cases appeared smaller and shrunken compared in Fig. 1 and scraped off, and the cells from four or five 90-mm dishes with that in intact cells. It was evident from time course observations including the supernatants were centrifuged at 5000 X g at4Â°Cfor 15 min. The that these cells lose their chromatin gradually, finally appearing as sediment was resuspended in 10.5 ml 50 mM Tris-HCl, 1 mM EDTA. and 1% chromatin-free "ghosts." Another type of apoptotic chromatin was SDS (pH 7.4) and incubated for 1 h at 37Â°Cwith 500 /il proteinase K solution also observed (not shown), namely, that of highly condensed nuclei in (10 mg/ml in PBS). Then, the homogenate was extracted sequentially with which the chromatin frequently is half-moon shaped without being equal volumes of phenol, a 1:1 mixture of phenol:chloroform:isoamyl alcohol scattered into smaller pieces. Both types were counted and summa (24:1), and chloroform:isoamyl alcohol (24:1). Three hundred jj.17.5 M aque rized as apoptotic nuclei. ous ammonium acetate solution were added to the supernatant of the last When TGF-ÃŸlwas added to rat hepatocytes plated at a density of extraction step, and 2 volume ice-cold ethanol were added. After storage at 100,000 cells/cm2, a concentration-dependent increase in the percent â€”¿20Â°Covernight, nucleic acids were sedimented completely by centrifugation at 5000 X g at 4Â°Cfor 10 min. The sediment was dissolved in 1 ml Tris-EDTA age of apoptotic nuclei was obtained 18 h later, i.e., 6 h after the and incubated for 3 h at 37Â°Cwith 125 /j.1 pancreatic RNase solution (10 second medium change (Fig. 3A). A very similar picture was obtained mg/ml in PBS). The DNA was then extracted by sequential phenol:chlorofom: with the carcinogenic aromatic amine 2-AAF (Fig. 3ÃŸ).The incuba isoamyl alcohol extraction and precipitated as described above. The sediment tion time of 12 h between the addition of 2-AAF and the second was washed twice in 70% ethanol. dried at room temperature, and dissolved in medium change was sufficient to metabolize 2-AAF completely, 100-150 fil Tris-EDTA. DNA concentration and purity were determined using which was confirmed by high-performance liquid Chromatographie the 260:280 nm ratio of A. Thirty p.g DNA/lane were loaded on an agarose gel analysis of the supernatants and cell extracts using highly sensitive (1% low-melting-point agarose and 10 /j.g/ml ethidium bromide in Tris-borate- fluorescence detection (not shown). 32P-postlabeling studies revealed EDTA). After 2-3 h of electrophoresis, gels were removed, and DNA was that overnight incubation of hepatocyte cultures with 50 /LIM2-AAF visualized using a UV transilluminator. led to the formation of DNA adducts (not shown). 2-AAF concentra Immunoprecipitation and Western Analysis. Cells were treated with UV tions greater than 50 /AMresulted in overt cytotoxic effects monitored light as shown in Fig. 1, washed with ice-cold PBS, scraped off. incubated for as leakage of lÃ¡clatedehydrogenase into the medium and were not 30 min in 500 /xl extraction buffer [10 mM Tris-HCl, 140 mM NaCl, 3 mM used in further experiments. UV irradiation also resulted in a dose- MgCl,, and 0.5% Nonidet P-40 (pH 7.0)] under repeated agitation, and dependent, significant increase in apoptotic nuclei, which reached a centrifuged at 10,000 x g. The supernatant (extract) was analyzed for protein, maximum at 150 J/m2 (Fig. 3C). and equal amounts of protein were incubated overnight with 1-5 /xg primary antibody (monoclonal anti-p53). The immunocomplex was then sedimented by In a subsequent series of experiments, the hypothesis was tested centrifugation (10.000 X g) at room temperature, washed twice in extraction that treatment with the liver tumor promoters TCDD and phÃ©nobar buffer and once in PBS, dried, suspended in 50 /Â¿ILaemmli buffer, heated for bital modifies the apoptotic response of the culture. It was found that 10 min at 65Â°C,and separated by PAGE. The proteins were electroblotted to neither TCDD nor phÃ©nobarbitalhad any suppressing effect on apop- a polyvinylidene difluoride membrane (Milipore, Dreieich. Germany), and the tosis induced by TGF-ÃŸl (Fig. 4). An identical result was obtained antigen was visualized with a primary antibody (monoclonal anti-p53) and a when TGF-ÃŸl was not removed before adding TCDD or phÃ©nobar secondary peroxidase-coupled, antimouse IgG antibody using the Amersham bital (not shown). At time points a96 h, however, when the apopto (Braunschweig. Germany) enhanced chemiluminescence protocol. genic effect of TGF-ÃŸlwas no longer observable, 2 mM phÃ©nobarbital Statistical Methods. For multiple comparisons (with an untreated control), and 1 nM TCDD suppressed the rate of background apoptosis also the Dunnett test procedure was used, applying SAS software (SAS, Inc., Gary, obtained in untreated cultures. NC). P = 0.05 was allowed for significance. In contrast to TGF-ÃŸl-induced apoptosis, the addition of 1 nM TCDD 12 h after 2-AAF treatment abrogated the wave of apoptosis observable without TCDD almost completely at 18 h (Fig. 5). A very Platingii1-15li-15!-15Med.changeiIl-12ÃŽTGF-B1J-12ÃŽ2-AAFj-12Med.change10 similar picture was obtained when 2-AAF-pretreated hepatocytes were treated with phÃ©nobarbital (Fig. 6). PhÃ©nobarbital also sup 1*t1 pressed apoptosis significantly at later time points, an effect that was ?of less pronounced with TCDD. Interestingly, phÃ©nobarbitalshowed this DetectionÃŽ+/- apoptoticnuclei1 effect both in untreated and in 2-AAF-treated cultures. When the cultures were treated with a UV dose of 90 J/m2. 1 nM promoter_ TCDD or 2 mM phÃ©nobarbitalalso reduced the incidence of apoptosis _ !..10 >of 1i1 significantly 6 and 12 h after medium change (Fig. 7). In addition to ,DetectionÃŽ+/- apoptotlcnucleil the morphological detection of apoptotic nuclei, internucleosomal fragmentation of DNA (laddering) was analyzed after UV treatment. promoter! At the high density used, a certain amount of background fragmen tation was observed (Figs. 8 and 9). Twenty-four h after irradiation 1. ..1 *>t l 1-0.5 â€¢¿*of 1 with 90 or 120 J/m2, a marked increase in fragments was obtained. 0 Detection1 apoptotlc nuclei When 1 nMTCDD (Fig. 8) or 2 mM phÃ©nobarbital(Fig. 9) were added */- promoter Time (h) 30 min after irradiation, however, the increase in DNA fragmentation was blocked almost completely. Interestingly, the addition of 1 nM Fig. 1. Regimen of application of apoptosis-initiating agents and tumor promoters to TCDD without prior UV treatment resulted in a slight increase in rat hepatocytes in primary culture. DNA fragmentation. Treatment with TGF-ÃŸlor 2-AAF led to a much 1273

Fig. 2. Apoptotic hepalocyte {arrow} in a pri mary culture of ral hepatocytes treated with 20 Â¡JL\\ 2-AAF for 12 h. Chromalin is visualized with Hoechst H33528. Upper Â¡tunel,visible and fluores cence light; lower panel, fluorescence light.

lower degree of DNA fragmentation (not shown), which hampered the Bursch et al. (9, 19). The authors demonstrated that enzyme-altered analysis of the effects of the promoting agents. preneoplastic rat liver foci showed a higher rate of not only prolifer Immunoprecipitation of the tumor suppressor gene product p53 ation but also of apoptosis. Furthermore, withdrawal of the liver tumor from total cell homogenates revealed a dose-dependent increase 24 h promoters phÃ©nobarbitalandcyproterone acetate from animals bear after UV treatment (Fig. 10). In agreement with the DNA fragmen ing preneoplastic livers resulted in a pronounced increase in apoptosis tation experiment, the addition of 1nMTCDD 30 min after irradiation in preneoplastic foci (9). These findings led to the suggestion that abrogated the UV-dependent increase in p53 almost completely. At the 0- and 90-J/m2 levels, however, 1 n.MTCDD resulted in a slight suppression of apoptosis may be an important mechanism of action of liver tumor promoters. increase in p53. In heputocyte cultures, the basal incidence of apoptosis seems to be slightly higher than in vivo. The exact reasons for this difference are DISCUSSION currently unknown. However, apoptosis in hepatocyte cultures can be In the past two decades, the study of liver tumor promotion in induced strongly by various factors, such as genotoxic agents, cyto- laboratory rodents contributed considerably to our current understand kines (e.g., TGF-ÃŸl),and growth factor deprivation (20, 21). TGF-ÃŸl ing of the principles of carcinogenesis. The concept of apoptosis as an also acts as a strong inhibitor of proliferation of most epithelial, important determinant in the multistage process of carcinogenesis endothelial, and hematopoietic cells (22). Although the signal trans- (1-3) was introduced into the field of liver tumor promotion by duction of TGF-ÃŸlis not yet established fully, its activity is depend- 1274

10 10 B

Â« 6

Fig. 3. Induction of apoptosis in rat hepatocytes in primary culture, treated at -12 h (see Fig. 1) with various concentrations of TGF-ÃŸl (A) or 2-AAF (B) or treated at O h with various doses of 0.1 0.2 0.5 1.0 2 5 10 20 50 UV light (C). Six h after medium change, apoptotic [TGF-B1] (ng/ml) [2-AAF] (MM) nuclei were identified using Hoechst H33528 stain 10 ing and fluorescence microscopy. Bars, means Â±SD of four independent experiments com prising three countings of 10' nuclei each. *. mean values significantly different (P ^ 0.05) from the untreated control.

0 60 90 120 150 200 UV light (J/m2)

Saline/DMSO DMSO/DMSO TGF-ÃŸ1/DMSO 2-AAF/DMSO

TGF-ÃŸl/2 mM phÃ©nobarbital DMSO/ 1nM TCDD TGF-B1/1 pM TCDD 2-AAF/ 1nM TCDD TGF-B1/1 nM TCDD

48 96 144 192 12 48 72 114 192 Incubation time (h) Incubation time (h)

Fig. 4. Effects of TCDD and phÃ©nobarbitalon the frequency of apoptosis induced in rat Fig. 5. Effects of TCDD on the frequency of apoptosis induced in rat hepatocytes in hepatocytes in primary culture with TGF-/3I. The percentage of apoptotic nuclei was primary culture with 2-AAF. The percentage of apoptotic nuclei was determined in cells determined in controls (saline/DMSO) 12 h after addition of 200 pg/ml TGF-/31. medium after treatment over 12 h with DMSO. medium change at 0 h. and addition of DMSO change (at 0 h). and subsequent treatment with DMSO (TGF-ÃŸl/DMSO). 2 mM phÃ©no (DMSO/DMSO) or 1 nM TCDD (DMSO/1 nu TCDD) or after treatment over 12 h with barbital (TGF-ÃŸl/2 mu phÃ©nobarbital). 1 pM TCDD (TGF-ÃŸl/l pM TCDD). or 1 nM 20 /Â¿M2-AAF,medium change at 0 h, and addition of DMSO (2-AAF/DMSO) or 1 nM TCDD (TGF-ÃŸl/l nu TCDD). Bars, means Â±SD of four independent experiments TCDD (2-AAF/l nM TCDD). Bars, means Â± SD of four independent experiments comprising three countings of IO1 nuclei each. *. mean values significantly different comprising three countings of IO1 nuclei each. * mean value significantly different (P (P ==0.05) from that of the TGF-/31/DMSO treatment. <0.05) from that of the 2-AAF/DMSO treatment. 1275

ent on membrane receptors. Receptor activation is thought to result in a decrease in cyclin-dependent kinase 4 activity, leading to growth arrest. Whether this pathway plays a role in TGF-/31-induced apop-

I | DMSO/saline â€¢¿â€¢2-AAF/saline UHI DMSO/ 2 mM phÃ©nobarbital 2-AAF/ 2 mM phÃ©nobarbital

I 60 60 90 90 120 120

Fig. 8. Effects of TCDD on internucleosomal DNA degradation in rat hepatocytes in primary culture. Hepatocyte cultures were treated with UV doses of 0, 60, 90, and 120 J/m2 as indicated (upper line), and DMSO (- ) or 1 nMTCDD ( + ) was added (lower Â¡ine). After 24 h, cells were harvested, and DNA was isolated and analyzed by agarose gel electrophoresis. Each lane contained 30 /xg DNA. DNA fragments were visualized with ethidium bromide and fluorescence. 0 6 12 48 72 114 192 Incubation time (h)

Fig. 6. Effects of phÃ©nobarbitalon the frequency of apoptosis induced in rat hepatocytes in primary culture with 2-AAF. The percentage of apoptotic nuclei was determined in cells after treatment over 12 h with DMSO. medium change at 0 h. and addition of saline (DMSO/saline) or 2 mM phÃ©nobarbital (DMSO/2 HIM phÃ©nobarbital) or after treatment over 12 h with 20 /UM2-AAF, medium change al O h, and addition of saline (2-AAF/saline) or 2 mM phÃ©nobarbital(2-AAF/2 WM phÃ©nobarbital).Bars, means Â±SD of four independent experiments comprising three countings of IO3 nuclei each. *. mean values significantly different (P s 0.05) from the corresponding control, i.e.. DMSO/ saline or 2-AAF/saline, respectively.

1 1DMSO1â€¢i

UV/DMSOUV/ phÃ©nobarbital"2 mM 3 -r--r////\UV/1nMTCDDiI*i1**

2 - Fig. 9. Effects of phÃ©nobarbitalon internucleosomal DNA degradation in rat hepato iKNSXSNNSNSNNNSNNNSNNlJL*1cytes in primary culture. Hepatocyte cultures were treated with UV doses of 0, 60, 90. and 120 J/m2 as indicated (upper line), and saline ( â€”¿)or 2 HIMphÃ©nobarbital( + ) were added (lower line). After 24 h, cells were harvested, and DNA was isolated and analyzed by agarose gel electrophoresis. Each lane contained 30 fig DNA. DNA fragments were visualized with ethidium bromide and fluorescence. 1 - P53 > â€¢¿' â€”¿ â€”¿â€¢

TCDD, lO-'M: - + + UV dose (J/m2): O O 90 90 120 120 150 150 6 12 24 Fig. 10. Western blot of immunoprecipitated p53 from homogenates of ral hepalocyte cultures 24 h after treatment with various UV doses as indicated. Thirty min after Incubation time (h) irradiation, DMSO (â€”¿)orI nMTCDD (+ ) was added. P53 was immunoprecipitated from equal amounts of homogenized cellular protein using monoclonal anti-p53 antibodies. Fig. 7. Effects of TCDD and phÃ©nobarbitalon the frequency of apoptosis induced in rat hepatocyles in primary culture with UV light. Cells were treated with UV light (90 J/m2), Representative blot of three blots. and the percentage of apoptotic nuclei was determined after 30 min (O h) and at later time points as indicated. Directly after irradiation, DMSO (UV/DMSO), 2 mM phÃ©nobarbital (UV/2 mM phÃ©nobarbital),or I nM TCDD (UV/l nM TCDD) was added. Controls were tosis, however, remains to be determined. In rat hepatocytes. TGF-/3I treated with DMSO only (DMSO). Bars, means Â±SD of four independent experiments comprising three countings of IO3 nuclei each. *, indicate mean values significantly is a potent initiator of apoptosis. which reaches a maxmium reportedly different (P < 0.05) from that of the UV/DMSO treatment. 48-96 h after the addition of various concentrations to the cells (18). 1276

In our experiments, a very similar concentration-response relationship regulation of p53. Because TCDD was shown to enhance the phos was found for TGF-ÃŸl. phorylation of a variety of proteins via activation of protein kinases As DNA-damaging factors, the carcinogenic aromatic amine linked to the dioxin receptor (37), TCDD may act via a similar 2-AAF and UV irradiation were used. The results demonstrate that mechanism. Alternatively, TCDD may influence the expression of 2-AAF and UV light are potent inducers of apoptosis in rat hepato- genes involved in apoptosis. Because plating of hepatocytes at low cytes. It cannot be excluded, however, that other than DNA-damaging density results in a marked increase in p53,4 the comitogenic effect of effects of 2-AAF or UV light, e.g., on mitochondria! or other proteins TCDD with epidermal growth factor found in hepatocyte cultures of affecting intracellular free Ca2+ (23) or other signaling pathways, are low density (38) may be related to a restriction of this increase. involved in the initiation of apoptosis in our experiments. An interesting observation in our experiments was that TCDD per In addition to characteristic morphological alterations in the nu se seemed to enhance the basal incidence of apoptosis in hepatocytes, cleus, internucleosomal DNA fragmentation is thought to be a hall an effect observed both in the chromatin-staining assay in cell cultures mark of apoptosis (24, 25). In cultured rat hepatocytes, it has been with 1 pin and in the DNA fragmentation assay with 1 nM. Whether the observed spontaneously (26) and after treatment with microtubule finding of a slight increase of the background level of p53 is related antagonists (23) but not after treatment with TGF-ÃŸl(18). Our results to an undefined genotoxic effect of TCDD or to an interference with confirm that the incidence of apoptotic nuclei observed microscopi p53 increasing signaling pathways remains to be elucidated. cally is not related to the degree of DNA fragmentation strictly. Our results demonstrate that the liver tumor promoters TCDD and Although UV irradiation resulted in a high yield of fragmented DNA, phÃ©nobarbitalsuppressapoptosis induced in rat hepatocytes by treatment treatment with TGF-ÃŸl and also with 2-AAF led to a much lower with 2-AAF or UV light significantly. In contrast, TGF-ÃŸl-induced amount of DNA fragments detectable. This difference may be related apoptosis was not affected by the promoting agents. These findings to the time courses of chromatin condensation, fragmentation, inter suggest that prevention of a subtype of apoptosis initiated by DNA nucleosomal ladder formation, and complete degradation of DNA, damage is a major mechanism of action of TCDD and phÃ©nobarbital which need investigation in more detail. The findings emphasize that during liver tumor promotion. Furthermore, our findings indicate that more than one indicator of apoptosis should be used in studies on the TCDD suppresses the increase in p53 after apoptogenic UV treatment. effects of putative apoptogenic or apoptosis-suppressing compounds. Further work is necessary to investigate the underlying mechanism, i.e., In the case of UV irradiation, however, the pronouced suppression of the influence of TCDD and other liver tumor promoters on the half-life, DNA fragmentation with TCDD or phÃ©nobarbitalwas in complete phosphorylation state, and transcriptional activity of p53. agreement with the data obtained from morphological inspection of hepatocyte cultures. REFERENCES A major finding of this study is a significant decrease in apoptosis monitored as condensed or fragmented nuclei when the liver tumor 1. Williams. G. T. Programmed cell death: apoptosis and oncogenesis. Cell. 65; 1097- 1098, 1991. promoters TCDD and phÃ©nobarbitalwere added after apoptogenic 2. Evan, G. I., Wyllie, A. H., Gilben. C. S., Littlewood. T. D., Land, H.. Brooks, M., treatments with UV light or 2-AAF. Recently, Bayly et al. (27) Waters, C. M.. Penn, L. Z., and Hancock. C. Induction of apoptosis in fibroblasts by c-myc protein. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. Influence of Liver Tumor Promoters on Apoptosis in Rat Hepatocytes Induced by 2-Acetylaminofluorene, Ultraviolet Light, or Transforming Growth Factor β1

Wolfgang Wörner and Dieter Schrenk

Cancer Res 1996;56:1272-1278.

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