Mapping of DNA alkylation sites induced by aziridinylbenzoquinones in human cells by ligation-mediated polymerase chain reaction.

Abstract

Diaziridinylbenzoquinones such as 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its 2,5-methyl analog (MeDZQ) require bioreductive activation in order to elicit their cytotoxic activities. In this study, we have mapped the intracellular alkylation sites induced by DZQ and MeDZQ in a single copy gene at the nucleotide level using ligation-mediated polymerase chain reaction. We have performed this analysis in two human colon carcinoma cells, one proficient (HT-29) and one deficient (BE) in DT-diaphorase (DTD) activity. In the DTD-proficient HT-29 cell line, DZQ and MeDZQ were found to alkylate both 5'-(A/T)G(C)-3' and 5'-(A/T)A-3' sequences. This is consistent with the nucleotide preferences observed when DZQ and MeDZQ are activated by purified DTD to reactive metabolites capable of alkylating DNA in vitro (C-S. Lee, J. A. Hartley, M. D. Berardini, J. Butler, D. Siegel, D. Ross, and N. W. Gibson. Biochemistry, 31: 3019-3025, 1992). Surprisingly in the DTD-deficient BE cell line a pattern of alkylation induced by DZQ and MeDZQ similar to that observed in the DTD-proficient HT-29 cells was observed. This suggests that reductive enzymes other than DTD can be involved in activating DZQ and MeDZQ to DNA-reactive species in vivo.