Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen

Background/Aims: Hepatocytes immortalized with a temperature-sensitive SV40 large T antigen (SV40Tag) function as well as primary hepatocytes followin g transplantation to reverse hepatic encephalopathy and improve survival in rodents with liver failure. The continued presence of SV40Tag in the condi tionally immortalized hepatocytes may increase the risk of malignant tumor growth in transplant recipients. Methods: We immortalized hepatocytes using a recombinant retrovirus contain ing the gene encoding SV40Tag flanked by loxP recombination target sites, E xcision of SV40Tag from immortalized cells could then be accomplished by si te-specific recombination with Cre-recombinase. Results: Cells immortalized with this recombinant virus expressed SV40Tag a nd doubled in number every 48 h, After excision of the gene encoding SV40Ta g with Cre-recombinase, cells stopped growing, DNA synthesis fell by 90%, a nd production of liver-specific mRNAs was either increased or became newly detectable. In addition, the morphology and epithelial cell polarity of the cells became more characteristic of differentiated hepatocytes. To determi ne their malignant potential, immortalized hepatocytes were transfected to express a second oncogene, activated H-ras. SV40Tag(+)/H-ras(+)-immortalize d cells were capable of anchorage-independent growth and developed into tum ors when injected in severe combined immunodeficiency mice. While Cre-recom binase delivery by recombinant adenovirus infection was not 100% efficient, when SV40Tag excision occurred anchorage-independent growth stopped and tu mor formation in immunodeficient mice was abolished. Immortalized hepatocyt es also contained the gene encoding herpes simplex virus thymidine kinase a nd treatment with ganciclovir produced complete regression of established t umors in mice. Conclusions: These studies extend previous work that indicates that a trans plantable hepatocyte cell line could be developed for clinical use.