Differential sensitivity of human and mouse alkyltransferase to O super(6)-benzylguanine using a transgenic model

O super(6)-benzylguanine (O super(6)-bG) potentiates nitrosourea cytotoxicity of human tumor xenografts in nude mice by inactivating O super(6)-alkylguanine-DNA alkyltransferase (AGT). Recent reports dispute whether murine AGT, in cell-free systems, is less sensitive to O super(6)-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 super(6)-bG, we evaluated in vitro and in vivo models of O super(6)-methylguanine-DNA methyltransferase gene (MGMT) expression in the same genetic background. The 50% inhibitory concentration of O super(6)-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 O super(6)-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. O super(6)-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 O super(6)-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 Leu super(180) was associated with O super(6)-bG resistance. These studies provide strong evidence that inactivation of AGT both in vivo and in vitro by O super(6)-bG is species selective and impacts O super(6)-bG-mediated enhancement of BCNU toxicity. This may influence the therapeutic index of O super(6)-bG-BCNU combinations observed in human tumor xenograft-bearing mice.