SENSITIZATION OF HUMAN BREAST-CANCER CELLS TO CYCLOPHOSPHAMIDE AND IFOSFAMIDE BY TRANSFER OF A LIVER CYTOCHROME-P450 GENE

The cancer chemotherapeutic agent cyclophosphamide (CPA) and its isome r ifosfamide (IFA) are alkylating agent prodrugs that require metaboli sm by liver cytochrome P450 (P350) enzymes for antitumor activity. The therapeutic effectiveness of these oxazaphosphorines is limited by th e hematopoietic, renal, and cardiac toxicity that accompanies the syst emic distribution of liver-derived activated drug metabolites. Transfe r of a liver cytochrome P450 gene, CYP2B1, into human breast MCF-7 can cer cells is presently shown to greatly sensitize these cells to oxaza phosphorine toxicity as a consequence of the acquired capacity for int ratumoral CPA and IFA activation. Thus, CPA and IFA were highly cytoto xic to MCF-7 cells following stable transfection of CYP2B1 but exhibit ed no toxicity to parental tumor cells or to a beta-galactosidase-expr essing MCF-7 transfectant. This cytotoxicity could be appreciably bloc ked by the CYP2B1 inhibitor metyrapone. Cell cycle analysis revealed t hat CPA arrested the CYP2B1-expressing cells, but not CYP2B1-negative cells, at G(2)-M phase. A strong bystander cytotoxicity effect that do es not require direct cell-cell contact was mediated by CYP2B1-express ing MCF-7 cells on non-CYP2B1 cells. Intratumoral CYP2B1 expression co nferred a distinct therapeutic advantage when treating MCF-7 tumors gr own in nude mice with CPA, as revealed by a 15-20-fold greater in vivo cytotoxicity, determined by tumor excision/colony formation assay, an d by the substantially enhanced antitumor activity, monitored by tumor growth delay, for CYP2B1-expressing MCF-7 tumors as compared to CYP2B 1-negative control tumors. These enhanced therapeutic effects were obt ained without any apparent increase in host toxicity. To evaluate the extent to which a CPA/P450 gene therapy strategy may be generally appl icable to other tumor cell types, a replication-defective recombinant adenovirus carrying the CYP2B1 gene driven by the cytomegalovirus (CMV ) promoter Ad.CMV-2B1 was constructed and used to infect a panel of hu man tumor cell lines. Ad.CMV-2B1 infection rendered each of the cell l ines highly sensitive to CPA and IFA cytotoxicity, with substantial ch emosensitization seen at multiplicities of infection as low as 10. The CPA/P450 prodrug activation system may thus serve as a useful paradig m for further development of novel cancer gene therapy strategies that utilize drug susceptibility genes to significantly potentiate the ant itumor activity of conventional cancer chemotherapeutic agents.