Retroviral transduction of a mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to O6-benzylguanine plus 1,3-bis(2-chloroethyl)-1-nitrosourea.
Human CD34 cells express low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Gene transfer of the AGT gene, methylguanine DNA methyltransferase (MGMT), results in only modest BCNU resistance. Recently, an AGT inhibitor, O6-benzylguanine (BG), entered clinical trials. In preclinical studies, BG potentiated the cytotoxic effect of BCNU in tumors but increased toxicity to normal CD34 cells. We transferred a mutant MGMT containing a glycine-to-alanine mutation at position 156, resulting in marked resistance to BG, into Chinese hamster cells; the K562 cell line and human CD34 cells used the retroviral backbone MFG. In each instance, cells expressed increased AGT and were much more resistant to the combination of BG and BCNU than the parental cells or cells transduced with wild-type MGMT. Furthermore, the transduction efficiency in human CD34 cells was in excess of 70%, and the proportion of CD34 transduced cells resistant to the combination was > 30%. Thus, retroviral-mediated transduction of a mutant MGMT into CD34 cells appears to be an effective way to induce selective resistance to a drug combination designed to overcome a significant resistance mechanism to nitrosoureas in tumors.