Mapping of DNA alkylation sites induced by adozelesin and bizelesin in human cells by ligation-mediated polymerase chain reaction.
In this study, we have mapped the intracellular alkylation sites of adozelesin and bizelesin, two potent analogs of CC-1065, in individual genes at the single-nucleotide level. Human colon carcinoma cells were treated with adozelesin and bizelesin, and the position of adducts were mapped within the PGK-1 and p53 genes by means of ligation-mediated polymerase chain reaction. The monofunctional alkylating agent adozelesin was found to alkylate genomic DNA predominantly within 5'-(A/T)(A/T)A* sequences. Additional sites of alkylation were observed within 5'-(A/T)(G/C)(A/T)A* sequences; however, these were considered to represent sites of medium to low preference. Bizelesin, a bifunctional analog capable of both DNA monofunctional alkylation and DNA interstrand cross-link formation, was also found to alkylate 5'-(A/T)(A/T)A* sequences. Putative bizelesin DNA interstrand cross-link sites indicated that AT-rich sequences are preferred in the intervening sequence between the two cross-linked adenines. Both six- and seven-nucleotide regions were identified as putative sites of DNA interstrand cross-link formation with 5'-TTTTTTA*, 5'-TTTATCA* and 5'-GTACTAA* sequences being preferred. Non-adenine bases are not observed as potential intracellular sites of either DNA interstrand cross-linking formation or monofunctional alkylation. Thus, the patterns of alkylation induced by adozelesin and bizelesin in genomic DNA are similar but not identical to that observed in purified cell-free DNA.
Lee
C S
CS
School of Pharmacy, University of Southern California, Los Angeles, 90033.
Pfeifer
G P
GP
Gibson
N W
NW
D000477:Alkylating Agents / Q000494:pharmacology*; D001483:Base Sequence; D001572:Benzofurans; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology*; D053138:Cyclohexenes; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D000080890:Duocarmycins; D016158:Genes, p53; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured; D014508:Urea / Q000031:analogs & derivatives* / Q000494:pharmacology
D000477:Alkylating Agents; D001572:Benzofurans; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D000080890:Duocarmycins; D007211:Indoles; C060090:adozelesin; D014508:Urea; D004247:DNA; D010735:Phosphoglycerate Kinase; C072257:bizelesin