FIG. 8.

Combined toxicity of oncolysis and prodrug activation by Ad constructs with the FCU1 gene inserted via SA into the late transcription unit. Melanoma cells (SK-MEL-28, Mel624) or keratinocytes (HaCat) were infected with Ads at indicated titers or were mock-infected in triplicates. 5-FC or medium was added 2 days post-infection at 10 mM (SK-MEL-28, HaCat) or at 5 mM (Mel624). Cytotoxicity was determined 6 days post-infection by staining of surviving cells with crystal violet. Cell content was determined by measuring optical density at 595 nm, and cell viability was plotted in percentage of mock-infected cells that did not obtain 5-FC. Mean values of triplicates are shown. For clarity of presentation, SDs are not shown and were below 16.6%. In HaCat cells, cytotoxicity of Ad5TyrSsp_mFCU was significantly lower (p < 0.05) than for Ad5Ssp_mFCU at titers higher than 3 TCID₅₀ and in the presence of 5-FC. In contrast, cytotoxicity of Ad5TyrSsp_mFCU was significantly higher (p < 0.05) than for Ad5Ssp_mFCU in SK-MEL-28 and Mel624 at titers between 0.3 and 30 TCID₅₀ and 1 and 30 TCID₅₀, respectively, in the presence of 5-FC. Cell viability was significantly reduced by 5-FC (p < 0.05) for Ad5CMVFCU at 100 and 300 TCID₅₀ (HaCat), at titers higher than 1 TCID₅₀ (SK-MEL-28), or at 300 TCID₅₀ (Mel624); for Ad5SL at 30 and 100 TCID₅₀ (HaCat) or at 30 TCID₅₀ (SK-MEL-28); for Ad5Ssp_mFCU at titers >3 TCID₅₀ (HaCat), at 1–10 and 100 TCID₅₀ (SK-MEL-28), or at 10 and 30 TCID₅₀ (Mel624); and for Ad5TyrSsp_mFCU at 300 TCID₅₀ (HaCat), 0.3– 30 TCID₅₀ (SK-MEL-28), or 1, 3, 30, and 300 TCID₅₀ (Mel624).