To improve the effects of suicide genes on spontaneous canine melanoma, we studied the responses to herpes simplex virus thymidine kinase (HSVtk) and yeast Saccharomyces cereviseae cytosine deaminase/uracil phosphoribosyl transferase fusion protein (Ycd::Yuprt). HSVtk phosphorylates ganciclovir (GCV) that finally
stops DNA replication. Ycd catalizes the passage from 5-fluocytosine (5-FC) to 5- fluorouracil (5-FU) a thymidine analogue that interferes with DNA replication while Yuprt drives to the synthesis of 5-FUTP that inhibits rRNA and mRNA processing. Monolayers were lipofected with plasmids carrying the therapeutic genes and subsequently the different concentrations of pro-drugs were added. As evidenced by a fused green fluorescent protein/HSVtk, fluorescent cells could be detected for more than 6 days after lipofection and treatment with GCV. We found that serial dilutions (up to 10% of the initial concentration) of the therapeutic genes with the non-therapeutic Escherichia coli b-galactosidase did not correlate with loss of cytotoxicity, showing a strong bystander effect. In order to evaluate senescence, after treatment cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside (X-gal) each 24 h. Cell viability was done by the acid phosphatase assay (APH) in monolayer and spheroid and the efficiency in lipofection was determined as the percentage of cells stained blue with X-gal. Our encouraging results support further research on the combination of suicide genes for local treatment of melanoma tumors in companion animals. Keywords: lipofection, bystander, thymidine kinase, cytosine deaminase.