Protection of hematopoietic cells from O-6-alkylation damage by O-6-methylguanine DNA methyltransferase gene transfer: studies with different O-6-alkylating agents and retroviral backbones

Overexpression of O-6-methylguanine DNA methyltransferase (MGMT) can protec t hematopoietic cells from O-6-alkylation damage. To identify possible clin ical applications of this technology we compared the effect of MGMT gene tr ansfer on the hematotoxicity induced by different O-6-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazin e derivative temozolomide. In addition, various retroviral vectors expressi ng the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or te mozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus -based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) wa s demonstrated for all four drugs. In comparison to mock-transduced control s, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on av erage 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematop rotection various vectors expressing the human MGMT-cDNA from a murine embr yonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-M GMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 mug/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clin ical studies aiming at hematoprotection by MGMT gene transfer.