Retroviral Transfer of a Bacterial Alkyltransferase Gene into Murine Bone Marrow Protects Against Chloroethylnitrosourea Cytotoxicity

The chloroethylnitrosoureas (CENUs) are important antineoplastic drugs for which clinical utility has been restricted by the development of severe delayed myelosuppression in most patients. To investigate the potential of DNA repair proteins to reduce bone marrow sensitivity to the CENUs, we transferred the Escherichia coli ada gene, which encodes a M_r 39, 000 O⁶-alkylguanine-DNA alkyltransferase (ATase), into murine bone marrow cells by the use of a high-titer ecotropic retrovirus. The ada-encoded ATase is resistant to O⁶-benzylguanine (O⁶-BG), a potent inhibitor of the mammalian ATases, thus affording the bone marrow an additional level of protection against CENUs. In methylcellulose cultures, ada-infected hematopoietic progenitor cells were twice as resistant as uninfected cells to the toxic effects of l, 3-bis(2-chloroethyl)-l-nitrosourea (BCNU) following treatment with O⁶-BG. Although showing no obvious protective effects against leukopenia, overexpression of the bacterial ATase activity reduced the severity of anemia and thrombocytopenia in mice treated with O⁶-BG and BCNU. These effects, which were maximal at a BCNU dose of 12.5 mg/kg, were associated with improved survival when BCNU was given at this dose. At lower BCNU doses cytotoxicity was limited in both transduced and control mice, and at higher doses the protective effect was saturated due to cytotoxicity. These results suggest that ada gene therapy may be a feasible approach to amelioration of delayed myelosuppression following O⁶-BG plus CENU combination chemotherapy.