Adenovirus mediated cytosine deaminase gene transduction and 5-fluorocytosine therapy sensitizes mouse prostate cancer cells to irradiation.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Anello
R
R
Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, New York, USA.
Cohen
S
S
Atkinson
G
G
Hall
S J
SJ
D000256:Adenoviridae; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D043525:Cytosine Deaminase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005437:Flucytosine / Q000494:pharmacology*; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D009700:Nucleoside Deaminases / Q000235:genetics* / Q000494:pharmacology*; D011355:Prodrugs / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000473:pathology / Q000532:radiotherapy*; D011836:Radiation Tolerance / Q000187:drug effects*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D000963:Antimetabolites; D011355:Prodrugs; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase