Evaluating the effect of prodrug metabolism on the bystander effect in cancer gene therapy

Kinta Marguerite-Culton Serve

Suicide gene therapy for cancer offers a selective approach to eradicating tumor cells by using non-toxic prodrugs in conjunction with prodrug-activating enzymes expressed at tumor sites. The efficacy of suicide gene therapy is often limited by the lack of a suitable gene delivery system and by the low activity of suicide enzymes towards the prodrug. These limitations may be overcome by using mutant and/or fusion enzymes that display increased activity towards a prodrug or by using enzymes that elicit a strong bystander effect (BE). The BE refers to the capability of transfected cells to induce death in neighboring untransfected cells, typically through intercellular transfer of cytotoxic prodrug metabolites. The Escherichia coli or bacterial cytosine deaminase (bCD) converts the prodrug 5-fluorocytosine (5FC) to the chemotherapeutic agent 5-fluorouracil (5FU). When coupled with E. coli uracil phosphoribosyltransferase (bUPRT), the resultant fusion enzyme, bCD/UPRT, induces greater cytotoxicity compared to bCD alone. Several bCD mutants generated in our laboratory also increase cytotoxicity relative to bCD. One mutant, bCD1525, was coupled with bUPRT in an attempt to augment cell death. It was hypothesized that expression of this fusion enzyme, bCD1525/UPRT, would enhance 5FC metabolism, thus generating higher concentrations of cytotoxic metabolites. We predicted that altered metabolite concentrations would shift the BE mechanisms away from 5FU diffusion (the mechanism typically reported for the 5FC/bCD system). We anticipated different mechanisms, namely transfer of apoptotic vesicles and metabolite movement through gap junctions, to play a more central role in the BE of cells expressing fusion enzymes. To examine 5FC metabolism, we developed two novel high-performance liquid chromatography (HPLC) methods, one to quantify 5FC anabolic products produced by suicide enzymes, the other to quantify a 5FC catabolic product generated by endogenous mammalian enzymes. Additionally, we examined the contribution of apoptotic vesicles to the BE and assessed the ability of our cancer cell lines to transfer compounds through gap junctions. Taken together, these data provide a more complete understanding of the relationship between prodrug metabolism, cytotoxicity, and the BE. This knowledge will aid in future endeavors to improve enzymes for more effective suicide gene therapy.

Evaluating the effect of prodrug metabolism on the bystander effect in cancer gene therapy

Files and links (1)

Abstract

Metrics

Details