Detection of labile anthracycline-DNA adducts by real-time PCR.

Abstract

Barminomycin was employed as a model anthracycline that yields thermally stable drug-DNA adducts. Real-time PCR was utilized for the detection of these barminomycin-DNA adducts at drug levels as low as 100 nM in a cell-free assay system, with the lowest level of detection at approximately 20 nM. By contrast, doxorubicin-DNA adducts are heat labile and their levels were underestimated by conventional real-time PCR unless the DNA denaturation temperature was lowered by the addition of glycerol. Doxorubicin-DNA adduct levels of 5.5 per 10 kb were detected by real-time PCR (in the presence of 24% glycerol) following treatment with 0.5 microM doxorubicin (and 2 mM formaldehyde), considerably more sensitive than that detected by a gene-specific Southern-based procedure. Both the absolute fluorescence intensity in the linear PCR amplification range and the crossing point method provided useful dose-dependent estimates of adduct levels. The time required for a complete real-time PCR analysis of drug-induced adduct levels was approximately 40 min, and this may ultimately provide oncologists with a rapid means with which to monitor drug-DNA adduct levels in patients under treatment with anthracyclines. Responses to these drugs could be quickly and efficiently monitored in patients, thereby facilitating optimization of drug dosages as well as early detection of resistance to these agents.