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Mapping of DNA Alkylation Sites Induced by Aziridinylbenzoquinones in Human Cells by Ligation-mediated Polymerase Chain Reaction¹

School of Pharmacy and Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033 [C-S. L., N. W. G.], and Department of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010 [G. P. P.]

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.

¹ This work was supported by NIH Grant CA 59537.

² To whom requests for reprints should be addressed, at Department of Biochemistry, College of Natural Sciences, Yeungnan University, Gyongsan 712, Korea.

³ Present address: Cancer Research Group, Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340.

Received 12/ 6/93. Accepted 2/21/94.