DNA-DAMAGE BY ANTICANCER AGENTS AND ITS REPAIR - MAPPING IN CELLS AT THE SUBGENE LEVEL WITH QUANTITATIVE POLYMERASE CHAIN-REACTION

The quantitative polymerase chain reaction (QPCR)-based assay was used to measure DNA damage and repair to a small (523 bp) fragment of the single-copy human N-ras gene in K562 cells. Compared with previous met hods DNA preparation from treated cells and the subsequent detection o f the radioactive product were considerably simplified. The results de monstrated that and QPCR can be used to measure damage in a small gene segment, caused by cisplatin, nitrogen, and quinacrine mustards. Drug -DNA adducts produced by two novel minor groove binding, sequence-spec ific molecules (AT-486 and DSB-120) could be detected at physiological ly relevant concentrations of drug. For both cisplatin and nitrogen mu stard the concentrations required to cause damage in cells were higher than those needed to cause equivalent damage in isolated DNA. In cont rast both AT-486 and quinacrine mustard caused more damage at equimola r concentrations in cells than in isolated DNA. DSB-120, which is clos ely related to AT-486, was found to be 15-fold less effective than the latter at causing damage in treated cells despite similar reactivity with isolated DNA. Repair of damage caused by quinacrine mustard to th e same small gene fragment was found to proceed at a constant rate ove r 24 h. The and QPCR assay presented here is a simple quantitative met hod to measure damage and repair in subgene functional units such as p romoters, introns, and exons. (C) 1994 Academic Press, Inc.