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.

cis‐diamminedichloroplatinum(II)

tetrachloro(dl‐trans)‐1,2‐diaminocyclohexaneplatinum (IV)

unlabeled CDDP‐modified salmon testis DNA

unlabeled, unmodified salmon testis DNA