DETECT OF DOXORUBICIN AND MITOXANTRONE INDUCED DRUG-DNA ADDUCTS IN TUMOR CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our overall aim is to enhance the anticancer activity of doxorubicin, one of the most widely used anticancer drugs in clinical use today. We have recently shown that doxorubicin is activated by formaldehyde (supplied by formaldehyde-releasing prodrugs which themselves are in clinical trials as single agents), and this results in a dramatic increase of the level of doxorubicin-DNA adducts in tumor cells in culture, together with a synergistic interaction between the two drugs (Cutts et al., Cancer Res., 61, 8194, 2001), resulting in a greatly enhanced, topoisomerase II  independent form of cell death (Swift et al., Cancer Res., 66, 4863, 2006). Because the doxorubicin is bound covalently to DNA by the formaldehyde activation, it is no longer a substrate for active efflux (a common form of acquired resistance to doxorubicin) and we have demonstrated that formation of these adducts can largely overcome P-glycoprotein and topoisomerase II-mediated resistance to doxorubicin (Cutts et al., Oncol. Res.,15, 199, 2005). We currently measure doxorubicin-DNA adducts by two experimental procedures, both of which have limitations: (1)Total genomic adduct levels (adducts per 10 kb of DNA) are routinely determined from scintillation counting of 14C labeled doxorubicin (which remains complexed to DNA following isolation from drug-treated cells), but this procedure is extremely expensive and also lacks sensitivity, thereby necessitating higher drug doses than required for growth inhibition. (2) Gene-specific adduct levels can also be determined from the extent of apparent cross-linking of DNA following isolation of the DNA from drug-treated cells (Cutts et al., Cancer Res., 61, 8194, 2001), however this procedure is very laborious and suffers from a large loss of adducts during the many experimental steps involved in the Southern hybridization procedure. The use of AMS largely overcomes both of these limitations and provides three major advantages for measuring doxorubicin-DNA adduct levels: (1) a dramatic increase of sensitivity (estimated as at least a 10,000 fold greater sensitivity) that would enable us to detect adducts at clinical (and sub-clinical) drug doses (2) the ability to measure the rate of repair of doxorubicin-DNA adducts in cells (3) potential to be able to monitor drug-DNA adduct levels in biopsies (blood, solid tumor) of patients undergoing chemotherapy in order to optimize drug treatment schedules.