Mechanisms of promotion of hepatocarcinogenesis in vitro

To investigate the mechanisms of promotion of hepatocarcinogenesis by phenobarbital (PB), two lines of chemically initiated rat hepatocytes, designated 6/15 and 6/27, were established in vitro. These hepatocytes maintained some features of differentiation in vitro as they express hepatocyte-specific genes such as albumin, cytochrome P-450 IIB and glutathione S-transferase-P. They also exhibited phenotypes in vitro that resemble those of initiated hepatocytes in vivo. Their growth and viability were dependent on both PB and growth factors in fetal bovine serum (FBS). PB was required for colony formation at low cell density in serum-supplemented medium. At higher cell density PB enhanced cell proliferation and inhibited cell lysis. The growth-enhancing effects of PB on one line of initiated hepatocytes (6/27) could be replaced by either conditioned medium from PB-independent 6/27 hepatocytes or the hepatocyte mitogen, TGF-alpha. This observation suggests that PB may promote hepatocarcinogenesis by enhancing the expression of autocrine growth factors in altered hepatic foci. The viability of initiated hepatocytes also was dependent on factors contained in FBS, as cells died when deprived of FBS. PB modulated hepatocyte death under conditions of serum deprivation. In low serum medium, 6/27 hepatocytes died by apoptosis in the presence of PB and by necrosis in the absence of PB. Neither of the cell lines was tumorigenic when transplanted into liver of syngeneic recipients at early and intermediate passages. One line, 6/15, became tumorigenic during in vitro passaging, indicating that initiated hepatocytes were capable of progression to hepatocellular carcinoma in vitro. Genes which respond to serum and PB immediately after exposure were studied in a clone of 6/27 hepatocytes (C1) which were highly dependent upon PB for growth in vitro. Expression of c-myc and c-fos in 6/27 C1 hepatocytes was induced by fresh medium. PB had some synergistic effect with factors in fresh medium and FBS on induction of c-fos expression. PB alone appeared to have little effect on induction of growth-related genes or other genes in 6/27 C1 hepatocytes. These studies demonstrated that PB may stimulate the growth of at least some initiated hepatocytes during in vitro cultivation. PB appears to function as a co-mitogen under growth-factor-sufficient conditions to enhance the growth of initiated hepatocytes in vitro, and as a membrane-stabilizer to prevent cell death by necrosis. This model also demonstrated that there were multiple stages for an initiated hepatocyte to progress to cancer in vitro. Initiated hepatocytes progressed from PB-dependent extended-lifespan/enhanced growth variants to PB-responsive immortal lines and finally to PB-independent tumorigenic hepatocytes. This PB-dependent in vitro progression of initiated hepatocytes mimics the process of promotion of hepatocarcinogenesis in vivo.