Detection of proteins that recognize platinum-modified DNA using gel mobility shift assay.
Using a gel mobility shift assay, we have identified proteins, in the nuclear extracts of a human lung cancer cell line, that recognize cis-diamminedichloroplatinum(II) (cis-DDP, CDDP)-modified DNA. A 158-base-pair double-stranded DNA fragment, derived from pBR322 plasmid DNA, was modified by either CDDP, tetrachloro(dl-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) or trans-DDP (the stereoisomer of CDDP and clinically ineffective). These platinum drug-modified probes were incubated with nuclear extracts and analyzed by gel mobility shift assay. Proteins in the extracts selectively recognized the clinically active platinum-modified DNA fragment. No binding to the trans-DDP-modified DNA fragment was observed. These proteins may play a role in the cytotoxicity or in a DNA repair process.
Fujiwara
Y
Y
Pharmacology Division, National Cancer Center Research Institute, Tokyo.
Kasahara
K
K
Sugimoto
Y
Y
Nishio
K
K
Ohmori
T
T
Saijo
N
N
D002945:Cisplatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D009944:Organoplatinum Compounds / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009944:Organoplatinum Compounds; C034697:transplatin; D004247:DNA; D002945:Cisplatin; C049777:ormaplatin