Differential sensitivity of human and mouse alkyltransferase to O6-benzylguanine using a transgenic model.

O6-benzlguanine (O6-bG) potentiates nitrosourea cytotoxicity of human tumor xenografts in nude mice by inactivating O6-alkylguanine-DNA alkyltransferase (AGT). Recent reports dispute whether murine AGT, in cell-free systems, is less sensitive to O6-bG than the human AGT protein, raising the possibility that efficacy seen in the mouse host may not predict the therapeutic index observed in clinical trials. To establish whether mouse and human AGT have different sensitivity to O(6)-bG, we evaluated in vitro and in vivo models of O(6)-methylguanine-DNA methyltransferase gene (MGMT) expression in the same genetic background. The 50% inhibitory concentration of O6-bG for inactivation of mouse AGT was >10-fold higher than for the human protein in MGMT-transfected Chinese hamster ovary (CHO) cells. A dose of O6-bG, which inactivated human AGT, markedly sensitized human MGMT-transfected CHO cells to 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), whereas mouse MGMT-transfected CHO cells were much more resistant. O6-bG inactivation of AGT in vivo was studied in livers of human MGMT-transgenic mice expressing both human and mouse AGT. After a single dose of O6-bG i.p., the 50% inhibitory concentration of AGT was higher for mouse than for human AGT. To reconcile our finding with those of others, we sequenced the mouse MGMT cDNA and found that mutation of amino acid residue Leu180 was associated with O6-bG resistance. These studies provide strong evidence that inactivation of AGT both in vivo and in vitro by O6-bG is species selective and impacts O6-bG-mediated enhancement of BCNU toxicity. This may influence the therapeutic index of O6-bG-BCNU combinations observed in human tumor xenograft-bearing mice.