Gene-directed enzyme prodrug therapy: quantitative bystander cytotoxicity and DNA damage induced by CB1954 in cells expressing bacterial nitroreductase

Abstract

Clones of human colon carcinoma (WiDr), ovarian carcinoma (SK-OV-3), and Chinese hamster V79 cells expressing the nitroreductase enzyme (NR) from E. coli B were 52-, 225- and 177-fold respectively more sensitive to a 24-h incubation with the prodrug 5-(aziridin-1-yl)-2,4- dinitrobenzamide (CB1954) than the parent lines. The IC₅₀s of non-NR-expressing bystander cells were measured in the presence of differing proportions of NR-expressing cells. The shift in IC₅₀ was used to calculate a value for the bystander effect, termed the transmission efficiency (TE), which is the decrease in IC₅₀ due to bystander effect as a percentage of the maximum decrease possible. The percentage of NR-expressing cells for which the TE was 50%, (the TE₅₀) is a single datum of bystander efficacy. WiDr and V79 cell lines, had a similar TE₅₀ of approximately 2%. SK-OV-3 gave a lower value of 0.3%. These TE₅₀ correlate with concentrations of cytosolic NR activity, which is distinguished from endogenous DT diaphorase activity by kinetic differences. A novel method is described which enables both DNA crosslinks and drug-induced single-strand breaks to be simultaneously quantified in a sedimentation assay. Using this technique, bystander DNA damage was demonstrated in V79 cells, of approximately 50% of that in activator cells.