DIFFUSIBLE CYTOTOXIC METABOLITES CONTRIBUTE TO THE IN-VITRO BYSTANDEREFFECT ASSOCIATED WITH THE CYCLOPHOSPHAMIDE CYTOCHROME-P450 2B1 CANCER GENE-THERAPY PARADIGM

Tumor cells become sensitive to the inert prodrug cyclophosphamide (CP A) after transfer of the gene encoding cytochrome P450 2B1. This enzym e activates CPA into 4-hydroxycyclophosphamide, which ultimately degra des into acrolein and phosphoramide mustard, the anticancer and DNA-al kylating metabolite, It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the pr oliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells, Prodrug-activating gene therapy sche mes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In co ntrast, we find that CPA-sensitized, P450-expressing C6 glioma cells ( C6-P450) transfer cytotoxicity to nonexpressing cells by releasing dif fusible metabolites through the medium, A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cell s, and medium conditioned by these cells can kill untransfected cells with similar potency, This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated, In an ani mal model of intracerebral brain tumors, expression of the P450 2B1 ge ne within the neoplastic cells enhanced significantly the antitumor ef fect of CPA, even when it was administered systemically, This study sh ows that CPA/P450 2B1 gene therapy represents a novel tumor-killing st rategy that displays an expanded range of cytotoxic action both spatia lly and temporally within tumor cells and significantly potentiates th e anticancer action of CPA when administered i.v.