RETROVIRAL INSERTIONAL MUTAGENESIS AS A STRATEGY FOR THE IDENTIFICATION OF GENES ASSOCIATED WITH CIS-DIAMMINEDICHLOROPLATINUM(II) RESISTANCE

Expression of resistance to cis-diamminedichloroplatinum(II) (CDDP), one of the most effective chemotherapeutic drugs used to treat a variety of malignancies, remains a serious obstacle for improving cancer treatment. To study possible genetic mechanisms underlying the development of CDDP resistance, we have adopted the approach of retroviral insertional mutagenesis. An early-stage CDDP-sensitive human melanoma cell line, WM35, was infected with a defective amphotropic murine retrovirus (murine stem cell virus), and the pooled cells were subsequently selected for CDDP-resistant variants. Nine CDDP-resistant clones independently derived from murine stem cell virus infected WM35 cells were analyzed and it was found that five of these clones acquired an identical retroviral integration site, designated as CDDP resistance locus 1 (CRL-1), as revealed by isolation of retroviral flanking sequences. Furthermore, using the flanking sequence as probe, we have detected a 3.5-4.0-kilobase message, the expression of which is strongly increased in clones carrying a rearranged CRL-1 locus. These results strongly suggest that overexpression of CRL-1 confers resistance to CDDP in these clones. In addition, the present study indicates that retroviral insertional mutagenesis represents a potential strategy to identify genes responsible for CDDP resistance and possibly other chemotherapeutic drugs as well.