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

Abstract

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.

Fig. 1a,b.

Histological and histochemical features of apoptoses in mouse liver. Representative micrographs of apoptoses in the liver of B6C3F1 mice are shown. a hematoxylin & eosin-stained liver sections, (↑) intracellular apoptotic body; note dense, homogenous, and eosinophilic cytoplasm with highly condensed chromatin, (↑↑) extracellular apoptotic bodies with and without visible chromatin, isolated from neighboring cells; b DNA fragmentation detected by the TUNEL assay in intracellular apoptotic bodies (↑↑); the TUNEL assay was performed as described in detail elsewhere (Grasl-Kraupp et al. 1995). Bar 10 μm

Fig. 2.

Incidence of apoptosis in normal and neoplastic mouse liver. NL phenotypically normal liver tissue, HCA hepatocellular adenoma, HCC hepatocellular carcinoma, n.f. not found in this subgroup of animals. NDEA (open columns); NDEA + 200 μg TGFβ1/kg (crosshatched columns); NDEA + PB (diagonally hatched columns); NDEA + PB + 200 μg TGFβ1/kg (columns hatched horizontally). Means are given; note that vertical bars indicate 95% confidence limits. Numbers at the columns number of lesions/numbers of cells scored