Identification of the SV40 Gene Product(s) Required for Induction of Chromosomal Aberrations in Human Cells

Abstract

Expression of the Simian Virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. The results of this study demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids either lacking the SV40 origin of DNA replication, or encoding a large T mutant defective in its ability to bind the retinoblastoma gene product (Rb) were as proficient as wild type plasmids, indicating that both viral DNA replication and binding of T antigen to Rb are not required for cytogenic damage. On the other hand, preliminary results with a plasmid encoding a T antigen mutant unable to bind the cellular p53 protein suggest that formation fo this complex may be important for cytogenetic damage. This study has also shown that chromosomal aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. These results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization.