CYTOSINE DEAMINASE::URACIL PHOSPHORIBOSYL TRANSFERASE/5-FC SUICIDE GENE SYSTEM DECREASES CLONOGENIC CAPACITY IN SURVIVING HUMAN MELANOMA CELLS

BERNABEI-CORNEJO, SOFIA; VILLAVERDE, MARCELA S; GLIKIN G. C.; FINOCCHIARO L.M.E

Congreso; SAIC 2022; 2022

The yeast cytosine deaminase::uracil phosphoribosyl transferase fusion protein associated to its prodrug 5-fluorocytosine (CDU/5FC), has been proposed as a suicide gene (SG) system for cancer therapy. Once expressed in the cell, CDU converts the 5FC prodrug in 5-fluorouracil (5-FU), which alters DNA and RNA processing and synthesis, leading to cell death. Previously, we have reported the cytotoxic effects of this system on eight human melanoma derived cell lines (A375, hM1, hM2, hM4, hM9, hM10, SB2 and M8). Five of these cell lines where derived from surgically excised human melanoma tumors, established and characterized in our laboratory (hM1, hM2, hM4, hM9 and hM10). We have shown that lipofection with CDU/5FC elicited a significant 5FC concentration-dependent cytotoxic response in these cell lines, compared to control lipofected cells. Even at the highest prodrug concentrations tested (1 mM 5FC), there were some remaining viable cells. In a clonogenic assay on A375, M8, hM4 and hM9 cell lines, we tested whether these remaining cells were still able to proliferate. We found that after 6 days of treatment of CDU- or control-lipofected cells, the clonogenic capacity of surviving cells was not altered at 1 µM, diminished at 10 µM and abolished at 100 µM 5FC. In parallel, dual acridine orange/ ethidium bromide (AO/EB) staining was performed at 24 h, 48 h and 96 h after treatment. Control cells mostly appeared as green-stained healthy cells. In CD::UPRT/5FC suicide gene treated cells, the number of late apoptotic/necrotic cells was significantly increased at 96 h, as evidenced by the increased rise of red-stained nuclei. We conclude that the CDU/5FC suicide system can significantly decrease human melanoma cells’ survival and that remaining cells are unable to proliferate.