ADENOVIRUS MEDIATED CYTOSINE DEAMINASE GENE TRANSDUCTION AND 5-FLUOROCYTOSINE THERAPY SENSITIZES MOUSE PROSTATE CANCER CELLS TO IRRADIATION

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Abstract
We assess the ability of adenovirus mediated expression of the Escherichia coli cytosine deaminase gene in conjunction with the prodrug 5-fluorocytosine to result in radiation sensitization in the mouse prostate cancer cell line RM-1 in vitro. To document cytotoxicity of gene therapy, RM-1 cells were exposed to escalating doses of adenovirus mediated cytosine deaminase and a fixed dose of 5-fluorocytosine or phosphate buffered saline. Viable cells as determined by exclusion of trypan blue were counted the following day. Cytosine deaminase expressing RM-1 cells were then irradiated as single cell suspensions at various doses of radiation in a cesium source (4.4 Gy. per minute) and randomized to receive 5-fluorocytosine therapy at different times in relation to the external radiation therapy. End points were determined in a clonogenic assay by counting colonies with greater than 50 cells 7 days after replating. Use of adenovirus mediated cytosine deaminase plus 5-fluorocytosine demonstrated viral dose dependent killing of RM-1 cells to a maximum of 85%, while either therapy alone was nontoxic. Neither adenovirus mediated cytosine deaminase infection nor 5-fluorocytosine alone influenced external radiation therapy killing. However, after controlling for death due to gene therapy alone, the combination of adenovirus mediated cytosine deaminase plus 5-fluorocytosine and external radiation therapy resulted in synergistic activity to approximately 2 logs of cell kill at low doses of radiation (p = 0.001). While altering the chronology of prodrug exposure in relation to external radiation therapy maintained synergy in all scenarios tested, starting 5-fluorocytosine 24 hours before external radiation therapy resulted in the most profound killing (p = 0.04), which indicates the importance of maintaining prodrug therapy during external radiation therapy. The combination of adenovirus mediated cytosine deaminase plus 5-fluorocytosine and radiation therapy resulted in radiation sensitization with clinically relevant doses of radiation suggesting a potential usefulness of this treatment in patients with prostate cancer.

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