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ras Transformation of Simian Virus 40-immortalized Rat Hepatocytes: An in Vitro Model of Hepatocarcinogenesis¹

Departments of Medicine [X-J. F., M. F., A. K., M. S.] and Pathology [R. C.], The Toronto Hospital and University of Toronto, Toronto, Ontario, Canada, M5G 2C4

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2–3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, -1-antitrypsin, fibrinogen, -1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.

¹ This study was supported by a grant from the Medical Research Council of Canada (MA 10175). M. F. is the recipient of a postdoctoral fellowship from the Medical Research Council of Canada. A. K. is a senior research scientist of the National Cancer Institute of Canada.

² To whom requests for reprints should be addressed, at Toronto General Hospital, En 9-225, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4.

Received 6/11/91. Accepted 10/22/91.