Treatment with dilute alkali-nuclease S1 permits the analysis of DNA damage: cells treated with platinum analogues.
We describe here an approach to characterize various lesions induced in DNA by drug treatments, using three parameters: (a) release of single-stranded DNA fragments by cell lysis in dilute alkali, which result from enzymatic strand scission during DNA repair or chemical alterations of DNA; (b) the presence of high molecular weight DNA in cells after lysis in dilute alkali followed by nuclease S1 treatment which, due to drug-induced DNA cross-links and its level is a measure of the amount of DNA-containing cross-links; and (c) the appearance of small double-stranded DNA fragments, when the cell lysis is followed by digestion with nuclease S1 to remove single-stranded DNA. This DNA shows the same characteristics as DNA of untreated cells, but it may contain monoadducts. By following the flow of label through the three parameters, one can characterize both the lesions induced in DNA and how the lesions are repaired. We report here results of three platinum analogues: cis-Pt(II), trans-Pt(II), and cis-FLAP(II). A large proportion of DNA in treated cells appears as fragments (parameter c). The cis- compounds and trans- compounds differ with regard to appearance of high molecular weight DNA (parameter b) and the initial release of fragments (parameter a).
D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology*; D003561:Cytarabine / Q000494:pharmacology; D004249:DNA Damage; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000187:drug effects; D004720:Endonucleases; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008545:Melanoma; D008795:Metronidazole / Q000031:analogs & derivatives* / Q000494:pharmacology; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013890:Thiourea / Q000494:pharmacology
D004273:DNA, Neoplasm; D009944:Organoplatinum Compounds; D003561:Cytarabine; D008795:Metronidazole; C034697:transplatin; C036789:dichlorobis(1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole)platinum (II); D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013890:Thiourea; D002945:Cisplatin