Conditional transformation of mouse liver epithelial cells. An in vitro model for analysis of genetic events in hepatocarcinogenesis.

Primary rodent and human hepatocytes have a very limited lifespan in culture and are not readily applicable to transformation studies in vitro. To facilitate the investigation of early genetic events involved in hepatocarcinogenesis, we examined a transformation assay system utilizing conditionally immortalized mouse liver epithelial cells as an alternative to primary hepatocytes. By infecting primary mouse hepatocytes with a recombinant retrovirus carrying a temperature-sensitive simian virus 40 large T antigen gene, two mouse liver epithelial cell lines, CHST8 and CHST10-2.1, were established. Because of the heat-labile nature of the large T antigen, the cell lines proliferated rapidly at 33 degrees C, but were growth-arrested at 39 degrees C. Because activated c-H-ras and c-myc oncogenes are frequently found to be involved in mouse hepatocarcinogenesis in vivo, we assessed whether those oncogenes can complement the immortalizing function of the large T antigen at the nonpermissive temperature. When CHST8 cells were doubly transfected with activated c-H-ras and c-myc at 33 degrees C, they exhibited clonal growth ability even after shifting the temperature to 39 degrees C. However, neither c-H-ras nor c-myc alone allowed growth at 39 degrees C. On the other hand, c-H-ras alone was sufficient for overcoming the growth defect of CHST10-2.1 cells at 39 degrees C, whereas c-myc alone was again ineffective. Northern blot studies revealed that endogenous c-myc expression was significantly downregulated in the parental CHST8 cells after a temperature shift from 33 to 39 degrees C. In contrast, in the parental CHST10-2.1 cells, appreciable c-myc expression was observed at both temperatures. These results indicate that c-H-ras and c-myc can cooperate in complementing the ability of the temperature-sensitive large T antigen to immortalize mouse liver cells at the nonpermissive temperature. In addition, the mutant c-H-ras, but not c-myc, cooperated with the functional T antigen at 33 degrees C to allow growth in soft agarose of the CHST8 and CHST10-2.1 cell lines. However, cell lines carrying mutant c-H-ras and overexpressing c-myc were unable to grow in soft agarose at 39 degrees C. Thus, the two cellular oncogenes were insufficient for full transformation of the liver epithelial cells. The present in vitro model should be useful for investigating molecular events involved in both early and late stages of hepatocarcinogenesis.