Establishment And Mechanistic Characterization Of SV40 T-Antigen Immortalized Human Fetal Liver Epithelial Cells

Hepatocellular carcinoma(HOC) is one of the most common cancers in the world. Epidemiological data indicated that the occurence of HCC is closely related to the persistent HBV infection and increased exposure to dietary aflatoxins. The precise molecular mechanism of hepatocarcinogenesis is still an enigma. Most investigations in this field have been performed on rodent hepatocyte systems. The study of human liver cells was done in primary cultures of limited numbers of cell divisions It was observed that rat hepatocytes could be transformed into an immortalized cell line after treatment with viral oncogenes, e.g. SV40 T gene. Efforts to immortalize normal human liver cells, however, were mostly unsuccessful. For this purpose, we have developed a serum-free medium system suitable for liver cell growth , and using this system , we had immortalized human fetal liver epithelial cells in vitro by retroviruses containing SV40 large T gene. The mechanistic characteristics of these cells in vitro SV40 T-immortalized fetal liver epithelial cells had also been characterized. The results of the studies were summarized as follows:1. A serum-free medium with increased level of hydrocortisone, plus some growth factors and hormones, was found to support the growth of human fetal liver epithelial cells for extented preiod of time . Meanwhile , the growth of fibroblasts was retarted .2. The viral titer from supernatants of G418-resistant PA317 cells transfected with pZpi(neo)SV40 T reached the level of 4 X 10~6 CFU/ml after one round of infection.3. Human fetal liver cells were first infected by SV40 T containing retroviruses in vitro. After 23 passages (195 days ) , the transformants entered the crisis stage. This period lasted for three months, and three of the four colonies survived. The surviving cells have been cultured over 40 passages, and are still well . Transformants were not found to be tumorigenic in nude mice for 4 months, indicating that these transformed liver cells are not malignant yet .4. The transformed cells showed the characteristics of liver epithelial cells in morphology , and clearly expressed cytokeratin 18 . These cells could also form "lobular "structure, but they did not express AFP when identified by immunocytochemistry.5. SV40 T was found in these transformed liver epithelial cells at levels of DNA. RNA and protein. The significant level of p53 as well as TGF-a proteins were demonstrated in these cells. These facts demonstrated that the immortalization of the cells was most likely due to the integration and the expression of SV40 T gene .The growth ability of transformants might be due to the cooperative effect of the inactivation of wild type p53, an important tumor suppressor gene in liver, as well as the activation of TGF-a, a critical oncogene in hepatocarcinogenesis...