Apoptosis in stages of mouse hepatocarcinogenesis: failure to counterbalance cell proliferation and to account for strain differences in tumor susceptibility.

C3H/He and B6C3F1 show much higher liver cancer susceptibility than C57BL/6J mice. We studied the hypothesis that this difference might result from failure of apoptosis. Hepatocarcinogenesis was induced by a single dose of N-nitrosodiethylamine (NDEA), followed by phenobarbital (PB) for up to 90 weeks. We observed (1) earlier appearance of putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) in C3H/He than in C57Bl/6J mice and (2) an increase of hepatocellular DNA synthesis in C3H/He and C57Bl/6J mice, compared to normal liver, via PPF and HCA to HCC. PB enhanced DNA synthesis and growth of PPF, in the C3H/He strain only, and of HCA and HCC of both strains. Apoptoses were rare in unaltered livers as well as in preneoplastic lesions, but tended to increase in HCA and HCC of both strains. PB lowered apoptotic activity in PPF of C3H/He mice, but enhanced it in HCA and HCC of C57Bl/6J mice at late stages. In conclusion, the strain difference in growth rates of PPF and tumors is largely determined by higher rates of cell proliferation in C3H/He mice, with and without promotion by PB. Moreover, in C57Bl/6J mice the promoting effect of PB was restricted to HCA and HCC and was not seen in PPF. Apoptosis was generally low and was not a major cause of the strain difference in tumor susceptibility. In contrast with rat liver, inhibition of apoptosis appears to be a minor determinant of tumor promotion in mice.

No increase of apoptosis in regressing mouse liver after withdrawal of growth stimuli or food restriction.

In short-term in vivo experiments, liver growth and regression in mice with high (C3H/He), intermediate (B6C3F1) or low (C57BL/6J) susceptibility to hepatocarcinogenesis was compared. Liver growth was induced by dietary administration of phenobarbital (PB; 750 ppm) or nafenopin (NAF; 500 ppm). PB or NAF treatment for 7 days produced moderate increases of liver DNA (15% or 25-28%, respectively) along with pronounced hypertrophy. Liver growth was strongest in C3H/He mice. Cessation of PB or NAF treatment led to a rapid regression of liver hypertrophy. However, the enhanced hepatic DNA content persisted for at least 2 weeks in all mouse strains. Apoptosis was not increased at any time after cessation of treatment in all strains. Food restriction to 60% of the ad libitum intake did not amplify either regression of liver hyperplasia or the occurrence of apoptosis. No strain difference in the occurrence of apoptosis was detected. Mouse hepatocytes in liver regressing after mitogen withdrawal do not enter apoptosis as readily as rat hepatocytes.

Induction of apoptosis in mouse liver adenoma and carcinoma in vivo by transforming growth factor-beta1.

PURPOSE: In the liver, transforming growth factor beta-1 (TGF-beta1) constitutes a major negative growth regulating factor involved in the control of cell numbers; failure of this control mechanism has been associated with the development of liver cancer. Since no reports on the in vivo effects of exogenously administered TGF-beta1 on apoptosis in liver tumors have been published yet, we studied hepatocyte sensitivity to the proapoptotic action of TGF-beta1 in stages of chemically induced mouse liver carcinogenesis. METHODS: Mouse liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w., i.p.) to 5-week-old B6C3F1 mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w) for 85 weeks. Four hours before being killed mice received a single dose of TGF-beta1 (56 microg or 200 microg TGF-beta1/kg of b.w., injected into the tail vein). Quantitative histological analysis of mitosis and apoptosis in normal liver tissue (NL), putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) was performed on H&E-stained liver sections. RESULTS: In NDEA and NDEA + PB-treated mice, NL exhibited a very low incidence of apoptosis and mitosis, which increased in HCA and HCC. In the lesions apoptoses ranged between 0.03 and 0.6%. Two hundred micrograms of TGF-beta1/kg stimulated apoptoses in NL as well as in neoplastic lesions (significant increase in NL, HCA, and HCC); the most pronounced proapoptotic action of TGF-ss1 was observed in lesions of NDEA+PB pretreated mice (about 1.7%). Fifty-six microg TGF-beta1/kg had no detectable effect on apoptosis. CONCLUSION: These observations indicate that during chemically induced liver carcinogenesis in B6C3F1 mice basal rates of apoptoses in adenoma and carcinoma are higher than in normal liver and can be further increased by a proapoptotic cytokine.