Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA

Abstract

Primary cultures of hepatocytes isolated by collagenase perfusion of adult rats were transformed by infection with adenovirus type 5 or transfection with adenovirus DNA. Total virion DNA or recombinant plasmid DNA containing the adenovirus EIA and E1B genes transformed hepatocytes at comparable frequencies. No foci of replicating hepatocytes were detected after transfection with a plasmid containing the ElA gene alone. The frequency of transformation by the adenovirus EIA and E1B genes was dependent on the composition of the culture medium. Transformation occurred at a low frequency when the transfected hepatocytes were maintained in a chemically defined medium (CDM), but the frequency was enhanced 8to 10-fold when the cells were maintained in (i) serum-supplemented medium or (ii) CDM supplemented with epidermal growth factor. Cell lines derived from the adenovirus-transformed colonies of hepatocytes expressed adenovirus EIA and E1B RNAs. When hepatocytes were maintained in CDM supplemented with dimethyl sulfoxide and transfected with plasmids containing the ElA and E1B genes, it was possible to derive cell lines that retained the ability to express several liver-specific genes, including albumin, transferrin, hemopexin, and the third component of complement. The amount of albumin secreted per cell varied from 1 to 5 pg per cell per 24 h, and in one cell line it was below detectable levels by passage 9. Adenovirus-transformed hepatocytes were not tumorigenic when inoculated subcutaneously into neonatal syngeneic rats. We conclude that the adenovirus ElA and E1B genes are capable of transforming adult rat hepatocytes, a differentiated epithelial cell type.

Expression of E-Cadherin and Other Paracellular Junction Genes Is Decreased in Iron-Loaded Hepatocytes

“…As the cultures age, no space remains between hepatocytes within the islands and paracellular junctions between adjoining cells can be observed by electron microscopy. 16,20 Primary rat hepatocytes in long-term DMSO culture have been used to study the molecular mechanisms of albumin expression, 17,21 altered growth control including induction of DNA synthesis and proliferation, 22 immortalization and transformation, [23][24][25] as well as programmed cell death. 26 More recently, we have demonstrated that hepatocytes in long-term DMSO culture function as an ideal model system for studying chronic iron loading of hepatocytes.…”

Strategies for immortalization of primary hepatocytes

“…Immortalized hepatocytes are typically derived from healthy primary hepatocytes by using a defined immortalization strategy. Both fetal and adult hepatocytes from different species have already been successfully immortalized, whether or not using a combination of viral oncogenes and the human telomerase reverse transcriptase (hTERT) protein [7, 9, 19-25]. The purpose of this paper is to discuss the different current immortalization strategies and provide an overview of the actually available immortalized hepatic cell lines and their applications.…”

Role of activated c‐H‐ras Oncogene in the induction and progression of immortal liver epithelial cell lines derived from normal C3H mice

“…In culture, neither transformation nor immortalization of primary rodent hepatocytes by activated rus transfection has been successful in our laboratories or in those of others, even when combined with active cellular replication induced by specific growth stimuli, although the simian virus 40 DNA or adenovirus DNA has been reported to be capable of immortalizing rat hepatocytes (Woodworth et al, 1986;Woodworth and Isom, 1987;Paul et al, 1988). This suggests that activation of the rus oncogene may not be an effective initiating event in the in vitro hepatocyte transformation system.…”