Medizinische Universität Wien, Univ. Klinik für Innere MedizinI, Abtl. Institut für Krebsforschung, Borschkegasse 8a, A-1090 Wien
1 To whom correspondence should be addressed. Fax : ++43 1 4277 9651. E-mail: wilfried.bursch{at}meduniwien.ac.at.
Received July 20, 2004; accepted February 14, 2005
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ABSTRACT |
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Key Words: phenobarbital; nafenopin; C3H/He; B6C3F1; C57Bl/6J; liver weight; protein content; DNA content; DNA synthesis; apoptosis.
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INTRODUCTION |
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In our studies on rat hepatocarcinogenesis, we have previously demonstrated that apoptosis constitutes a protective mechanism against cancer formation, and in 1984 we were the first to show that inhibition of apoptosis by liver tumor promoters accelerated the manifestation of frank neoplasia (Bursch et al., 1984; Schulte-Hermann et al., 1995
). However, relatively little is known about the role of apoptosis in determining susceptibility to liver cancer in different mouse strains. Based upon different long-term experimental models, somewhat conflicting data on apoptosis in mouse hepatocarcinogenesis have been reported (Goldsworthy and Fransson-Steen, 2002
; James and Roberts, 1996
; Kamendulis et al., 2001
; Sanders and Thorgeirsson, 2000
; Stevensson et al., 1999
).
Consequently, we addressed the question whether the high cancer susceptibility of B6C3F1 and C3H/He mice may result from low efficiency or even failure of cancer defense by apoptosis. In a first series of experiments we performed comparative short-term in vivo studies on liver growth (cell proliferation) and involution (apoptosis). C3H/He, C57Bl6/J, and B6C3F1 mice were treated with the "classical" tumor promoter phenobarbital (PB) or the peroxisome proliferator nafenopin (NAF) as model compounds. Short-term in vivo studies with mice and rats have been successfully used to characterize the liver response to drugs and industrial chemicals in view of their hepatocarcinogenic potency (for review, see Huber et al., 1996; Schulte-Hermann et al., 1995
; Whysner et al., 1996
). The studies addressing apoptosis regulation in normal mouse liver served to complement and to better understand the results of a long-term carcinogenesis study with these mouse strains (see Bursch et al., accompanying manuscript). The in vivo approach was chosen because the actual rates of cell replication and of apoptosis integrate survival- and death-controlling factors in the context of the organism's genetic background.
Here we report on the results of the short-term experiments (7- to 14-day PB or NAF treatment followed by withdrawal) focused on cell proliferation and apoptosis. The results revealed that PB/NAF-induced mouse liver growth largely is due to cell enlargement (hypertrophy). In all strains of mice under study, liver regression upon cessation of PB/NAF treatment was not associated with an increase in apoptoses; this observation was confirmed by biochemical analysis of liver DNA content. Furthermore, food restriction did not amplify liver regression and the occurrence of apoptosis. Thus, as to strain differences in cancer susceptibility, C3H/He mice exhibited a more pronounced DNA synthesis response to PB or NAF than C57Bl/6J, thereby revealing a positive correlation between short-term effects and cancer susceptibility. However, no difference among the mouse strains with respect to the occurrence of apoptosis was detected. Surprisingly, mouse hepatocytes do not appear to enter apoptosis as readily as rat hepatocytes (species difference).
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MATERIAL AND METHODS |
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DNA synthesis.
The rate of DNA synthesis was measured based upon BrdU-incorporation into DNA. For continuous infusion of BrdU (5-bromo-2'-deoxyuridine, Sigma Chemicals, St. Louis, MO) osmotic minipumps (Alza Corporation, Palo Alto, CA) were implanted sc under isoflurane anesthesia (Abbott Laboratories, Queensborough, UK; 1.5% isoflurane [v/v] in 60% N2O and 40% O2 [v/v]); 3-day pumps (Alzet model 1003D) and 7-day pumps (Alzet model 2001) were used to deliver a BrdU solution (20 mg BrdU/ml PBS) at a rate of 1 µl/h.
Sacrifice, biochemical, and histological procedures.
Animals were anaesthetized with isoflurane, decapitated, and exsanguinated. The liver was quickly excised, and the gall bladder was carefully removed. For the biochemical determination of hepatic DNA and protein content, representative liver samples from the large median lobe were frozen, stored in liquid nitrogen, and processed as described elsewhere (Grasl-Kraupp et al., 1994). Other liver specimens of the same lobe were cut into 4- to 5-mm thick slices and fixed either with Carnoy's solution or 4% neutral buffered formalin according to Lillie (Pearse, 1980
). Samples fixed in Carnoy's solution were transferred into isopropanol after 24 h and were stored in isopropanol until being processed for histology. Formalin-fixed tissue was processed after a fixation time of 24 to 48 h. Sections (3 to 5 µm) were mounted on APES-coated glass slides (3-aminopropyl-triethoxysilane, Fluka Chemie AG, Buchs, Switzerland) to ensure adhesion during processing. H&E (hematoxylin and eosin) staining was performed according to standard protocols. For the detection of BrdU-incorporation, formalin fixed liver specimens were processed as described in detail elsewhere (Chabicovsky et al., 2003
) using a mouse monoclonal antibody to BrdU (Boehringer-Mannheim, Mannheim, Germany; 1:800 in PBS/0.1% BSA). Biotinylated rabbit anti-mouse IgG (DAKO A/S, Glostrup, Denmark; 1:100 in PBS/0.1% BSA) was used as second antibody, followed by peroxidase-conjugated streptavidin (DAKO A/S, Glostrup, Denmark; 1:100 in PBS/0.1% BSA) and visualized with DAB (3,3'-diaminobenzidine tetrahydrochloride). Sections were counterstained with Mayer's hematoxylin and mounted with Entellan.
Labeling index and apoptotic index.
The rate of DNA synthesis was determined histologically in BrdU-stained liver sections; a section of duodenum, a tissue with a high cell proliferation rate, was included on each slide to confirm the systemic delivery of BrdU to the animal. At least 2000 hepatocyte nuclei per animal were counted using a Nikon Microphot-FXA microscope (Tokyo, Japan). The labeling index (LI) was expressed as percentage of labeled hepatocyte nuclei in the total population of nuclei counted. For quantitative determination of apoptosis, 30004000 hepatocytes per animal were scored. Hepatocytes exhibiting chromatin condensation typical of apoptosis, apoptotic bodies with or without chromatin located intra- or extracellularly were recorded. The total number of apoptoses was expressed as percentage of the total number of hepatocytes counted; the low apoptotic counts as determined by scoring 30004000 hepatocytes per liver section were verified by screening whole liver sections. The validity of this method for quantitative determination of apoptoses with H&E-stained liver sections has been demonstrated previously (Chabicovsky et al., 2003; Schulte-Hermann et al., 1995
).
Statistics.
If not stated otherwise, means (± SD) are given; data were analyzed by ANOVA, followed by Tukey-Kramer multiple comparisons test. The significance level was set at p < 0.05.
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RESULTS |
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Apoptoses were extremely rare at any time point after cessation of treatment in control and withdrawal groups of all mouse strains (Table 2). The lack of increased numbers of apoptoses in involuting livers of mice is in accordance with the persistent enhancement of liver DNA.
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DISCUSSION |
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Moreover, other in vivo as well as cell culture studies revealed that mouse hepatocytes are much less sensitive to the pro-apoptotic action of TGF-ß1 than rat hepatocytes (Chabicovsky et al., 2003; Parzefall et al., 2002
). Thus, 56 µg TGF-ß1/kg did not induce apoptosis in mouse liver, whereas in rat liver a dose as low as 0.25 µg TGFß-1/kg was found to be effective (Chabicovsky et al., 2003
; Oberhammer et al., 1992
; Schulte-Hermann et al., 2002a
; Fig. 6). Likewise, comparative studies with primary cultures of hepatocytes from C3H and C57BL mice revealed that at any concentration between 0.1 and 100 ng/ml TGF-ß1 exerted only a weak proapoptotic effect (less than 1%), whereas rat hepatocytes (F344) cultured under the same conditions were readily stimulated to undergo apoptosis at concentrations of 0.3 ng/ml or higher (up to 6%, ED50 = 0.8 ng/ml; Parzefall et al., 2002
; Fig. 6). Notably, mouse hepatocytes are highly sensitive to the pro-apopotic action of ligands of the TNF/NGF-receptor family (for review, see Kanzler and Galle, 2000
; Schulte-Hermann et al., 2002b
); it is tempting to raise the hypothesis that rats and mice might differ with respect to the relative contribution of various cytokine networks to control of hepatocellular apoptosis.
In conclusion, the present results show that the tumor-promoting efficiency of prolonged PB treatment corresponds to the short-term effects of PB in mouse liver. Thus, among the three inbred mouse strains studied, the highly cancer-susceptible C3H/He mice exhibited the strongest growth response to the tumor-promoting agent PB. Surprisingly, no strain difference with respect to the occurrence of apoptosis was found. This conclusion meets well with our observations in a long-term carcinogenesis study on C3H/He and C57Bl/6J mice, which indicates that the rate of cell proliferation largely determines susceptibility for tumorigenesis (see Bursch et al., accompanying manuscript). On the other hand, profound differences in the sensitivity to pro-apoptotic signals appear to exist between mouse and rat hepatocytes.
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NOTES |
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ACKNOWLEDGMENTS |
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