Mouse Mammary Tumor Virus Promoter‐Containing Retroviral Promoter Conversion Vectors for Gene‐Directed Enzyme Prodrug Therapy are Functional In Vitro and In Vivo

Reinhard Klein,Thomas Peterbauer,Bärbel Ruttkowski,Brian Salmons,Christine Hohenadl,Sonja Schwab,Walter H Günzburg

doi:10.1155/2008/683505

Abstract

Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

Highlights

Conventional cancer chemotherapy including chemotherapy of breast cancer often results in severe systemic toxicity at drug concentrations necessary for effective killing of tumor cells
293/pPCEMm1 + CRFK/DsRed 2GP19Talf/pPCEMm1 + CRFK/DsRed (b) expression of the eGFP reporter gene from an MLV promoter/CMV enhancer cassette while in infected cells after promoter conversion, expression of the eGFP gene is driven by the heterologous mammary tumor virus (MMTV) promoter
The promoter conversion (ProCon) vectors we have generated in this study are a further development of an earlier, a β-galactosidase reporter gene-containing vector that was used for the generation of virus-producing cells for encapsulation experiments [19]

Summary

Introduction

Conventional cancer chemotherapy including chemotherapy of breast cancer often results in severe systemic toxicity at drug concentrations necessary for effective killing of tumor cells. This obstacle can be overcome with the concept of gene-directed enzyme prodrug therapy (GDEPT) that implies selective delivery into tumor cells and expression of drug-metabolizing transgenes within them [1]. The oxazaphosphorine cyclophosphamide (CPA) and its structural isomer ifosfamide (IFA) are DNA-alkylating agents commonly used in breast cancer chemotherapy [2] These anticancer agents are administered as prodrugs that are primarily activated by the hepatic enzyme cytochrome P450 (CYP). Cyclophosphamide and ifosfamide suicide gene therapy has the advantage of exerting a bystander effect as it causes the death of Journal of Biomedicine and Biotechnology the therapeutic transgene-carrying cells and of neighboring nontransgenic cells via passive diffusion of the cytotoxic metabolites [5, 6]

Methods

Results

Conclusion