Mapping of DNA alkylation sites induced by aziridinylbenzoquinones in human cells by ligation-mediated polymerase chain reaction.
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.
Lee
C S
CS
School of Pharmacy, University of Southern California, Los Angeles 90033.
Pfeifer
G P
GP
Gibson
N W
NW
D000478:Alkylation; D000970:Antineoplastic Agents / Q000378:metabolism*; D001388:Aziridines / Q000378:metabolism*; D001483:Base Sequence; D016227:Benzoquinones / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D003110:Colonic Neoplasms; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D005091:Exons; D016158:Genes, p53; D006801:Humans; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001388:Aziridines; D016227:Benzoquinones; D017931:DNA Primers; D004273:DNA, Neoplasm; C073715:2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone; C009970:ethylenimine quinone; D004247:DNA; D016660:NAD(P)H Dehydrogenase (Quinone); D010735:Phosphoglycerate Kinase