GENE-DIRECTED ENZYME PRODRUG THERAPY - QUANTITATIVE BYSTANDER CYTOTOXICITY AND DNA-DAMAGE INDUCED BY CB1954 IN CELLS EXPRESSING BACTERIAL NITROREDUCTASE

Clones of human colon carcinoma (WiDr), ovarian carcinoma (SK-OV-3), a nd Chinese hamster V79 cells expressing the nitroreductase enzyme (NR) from E. coli B were 52-, 225- and 177-fold respectively more sensitiv e to a 24-h incubation with the prodrug 5-(aziridin-1-yl)-2,4-dinitrob enzamide (CB1954) than the parent lines. The IC(50)s of non-NR-express ing bystander cells were measured in the presence of differing proport ions of NR-expressing cells. The shift in IC50 was used to calculate a value for the bystander effect, termed the transmission efficiency (T E), which is the decrease in IC50 due to bystander effect as a percent age of the maximum decrease possible. The percentage of NR-expressing cells for which the TE was 50%, (the TE50) is a single datum of bystan der efficacy. WiDr and V79 cell lines, had a similar TE50 of approxima tely 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 meth od is described which enables both DNA crosslinks and drug-induced sin gle-strand breaks to be simultaneously quantified in a sedimentation a ssay. Using this technique, bystander DNA damage was demonstrated in V 79 cells, of approximately 50% of that in activator cells.