DRUG-RESISTANCE TO 5-AZA-2'-DEOXYCYTIDINE, 2',2'-DIFLUORODEOXYCYTIDINE, AND CYTOSINE-ARABINOSIDE CONFERRED BY RETROVIRAL-MEDIATED TRANSFER OF HUMAN CYTIDINE DEAMINASE CDNA INTO MURINE CELLS

Purpose: The hematopoietic toxicity produced by the cytosine nucleosid e analogs is a critical problem that limits their effectiveness in can cer therapy. One strategy to prevent this dose-limiting toxicity would be to insert a gene for drug resistance to these analogs into normal bone marrow cells. Cytidine (CR) deaminase can deaminate and thus inac tivate 5-aza-2'-deoxycytidine (5-AZA-CdR), 2',2'-difluorodeoxycytidine (dFdC) and cytosine arabinoside (ARA-C). The aim of this study was to determine if gene transfer of CR deaminase into murine fibroblast cel ls confers drug resistance to these cytosine nucleoside analogs and if this resistance can be prevented by the CR deaminase inhibitor, 3,4,5 ,6-tetrahydrouridine (THU). Methods: NIH 3T3 murine fibroblast cells w ere transduced with retroviral particles containing the human CR deami nase cDNA. Assays measuring CR deaminase activity as well as the inhib itory action of 5-AZA-CdR, dFdC and ARA-C on colony formation, were pe rformed in the presence of different concentrations of THU. Results: R etroviral-mediated transfer of the CR deaminase gene into 3T3 fibrobla sts produced a considerable increase in CR deaminase activity. The tra nsduced cells also showed significant drug resistance to 5-AZA-CdR, dF dC and ARA-C, as demonstrated by a clonogenic assay. This drug resista nce phenotype and elevated CR deaminase activity were reversed by THU. Conclusions: These findings indicate that the CR deaminase gene can p otentially be used in cancer gene therapy for protecting normal cells against the cytotoxic actions of different cytosine nucleoside analogs . In addition, the CR deaminase-transduced cells can be used as a mode l for screening different CR deaminase inhibitors in an intact cellula r system.