INTRATUMORAL ACTIVATION AND ENHANCED CHEMOTHERAPEUTIC EFFECT OF OXAZAPHOSPHORINES FOLLOWING CYTOCHROME-P-450 GENE-TRANSFER - DEVELOPMENT OFA COMBINED CHEMOTHERAPY CANCER GENE-THERAPY STRATEGY

Cyclophosphamide and its isomer ifosfamide are cell cycle-nonspecific alkylating agents that undergo bioactivation catalyzed by liver cytoch rome P-450 enzymes. The therapeutic efficacy of these oxazaphosphorine anticancer drugs is Limited by host toxicity resulting from the syste mic distribution of activated drug metabolites formed in the liver. Si nce tumor cells ordinarily do not have the capacity to activate oxazap hosphorines, we examined whether introduction into tumor cells of a cD NA encoding CYP2B1, a major catalyst of oxazaphosphorine activation, s ensitizes the cells to the cytotoxic effects of cyclophosphamide and i fosfamide. Here we show that 9L gliosarcoma cells stably transfected w ith a cDNA encoding rat CYP2B1 are highly sensitive to cyclophosphamid e and ifosfamide cytotoxicity as compared to parental 9L cells or 9L c ells transfected with an Escherichia coli beta-galactosidase gene. The CYP2B1 enzyme inhibitor metyrapone protects the CYP2B1-expressing 9L cells from oxazaphosphorine cytotoxicity, demonstrating that the chemo sensitivity of these cells is a direct consequence of intracellular pr odrug activation. Moreover, CYP2B1-expressing 9L cells potentiate the cytotoxic effects of cyclophosphamide and ifosfamide toward cocultured CYP2B1-negative 9L tumor cells. This ''bystander effect'' does not re quire cell-cell contact, and therefore may have the therapeutic advant age of distributing cytotoxic drug metabolites to a wide area within a solid tumor mass. In vivo experiments using Fischer 344 rats implante d s.c. with CYP2B1-expressing 9L tumor cells demonstrated that intratu moral expression of the CYP2B1 gene provides a substantial therapeutic advantage over that provided by liver cytochrome P-450-dependent drug activation alone; cyclophosphamide treatment resulted in complete gro wth inhibition of CYP2B1-positive tumors, whereas only a modest growth delay effect was obtained with CYP2B1-negative tumors. These studies establish that drug-activating CYP genes may be useful for the develop ment of novel combined chemotherapy/gene therapy strategies for cancer treatment utilizing established cancer chemotherapeutic agents.