Detection of platinum-DNA adducts by 32P-postlabelling.

摘要

We developed a sensitive 32P-postlabeling method for the detection of bifunctional intrastrand crosslinks d(Pt-GpG) and d(Pt-ApG) in DNA in vitro and in vivo. After enzymatic digestion of DNA the positively charged platinum adducts were purified from unplatinated products, using strong cation exchange chromatography. Subsequently the samples were deplatinated with cyanide, because platinated dinucleotides are very poor substrates for polynucleotide kinase. The excess of cyanide was removed using Sep-pak C18 cartridges, and the resulting dinucleoside monophosphates d(GpG) and d(ApG) were subsequently postlabelled. Analysis of the postlabelling mixture was performed by a combined TLC and HPLC-procedure. Good correlations with existing methods (AAS, immunocytochemistry and ELISA) were found in DNA samples treated in vitro and in vivo with cis- or carboplatin. The detection limit of the assay was 1 adduct/10(7) nucleotides in a 10 micrograms DNA sample.