Quenching of DNA cross-link precursors of chloroethylnitrosoureas and attenuation of DNA interstrand cross-linking by glutathione.

Interstrand DNA cross-linking is essential for the antitumor activity of chloroethylnitrosoureas (CENUs). The critical cross-links have been proposed to involve a rapid O6-guanine chloroethylation on one DNA strand, followed by a rearrangement of the O6-(2-chloroethyl)guanine and slow alkylation of the second DNA strand. In view of the relative intracellular abundance of glutathione (GSH) and nucleophilicity of its thiolate ion, the ability of GSH to react with and to inactivate 2-chlorethylated DNA and the possibility that this interaction decreases net DNA cross-linking by CENUs were investigated. Chloroethylated calf thymus DNA was reacted with GSH, the DNA was precipitated and redissolved, and subsequent DNA interstrand cross-linking was determined. The DNA cross-link index was compared for both GSH-treated and 2-chloroethylated untreated DNA. Simultaneously, Col E1 plasmid DNA was chloroethylated and reacted with GSH, and the extent of DNA interstrand cross-linking was determined by agarose gel electrophoresis and compared with controls. The results show both a time- and GSH concentration-dependent quenching of chloroethylated DNA, with a corresponding decrease in the DNA cross-link index. Using [methyl-3H]GSH, it was also demonstrated that 56% of the total GSH was bound to quenched 2-chloroethylated Col E1 DNA and 25% to quenched 2-chloroethylated calf thymus DNA. GSH binding to cross-linked DNA and native DNA was insignificant. It is concluded that, in addition to direct inactivation of reactive cytotoxic CENU species, GSH may also modulate cellular response to CENUs by quenching chloroethylated DNA, thereby decreasing the formation of potentially lethal DNA cross-links.

Duke Scholars

Author Francis Ali-Osman Neurosurgery, Neuro-Oncology