A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells.
A method for the determination of DNA monoadducts capable of forming interstrand crosslinks in mammalian cells is described. Such monoadducts were produced by brief treatment of cells with cis-diamminedichloro-Pt(II) (cis-DDP), 1-(2-chloroethyl)-1-nitrosourea (ClEtNU), L-phenylalanine mustard (L-PAM), or diaziridinylbenzoquinone (AZQ). The method is an alkaline elution procedure in which the DNA from lysed cells is incubated on polycarbonate filters at pH 10 and 37 degrees C. During this incubation, the progressive formation of interstrand crosslinking was observed in drug-treated cells. In the case of ClEtNU and AZQ, DNA strand breaks also formed, due to the presence of labile lesions in the DNA. This made quantitation of interstrand crosslinks difficult for these drugs. For cis-DDP and L-PAM, however, there was no significant production of strand breaks and the assay for interstrand crosslinks was quantifiable.
D001665:Binding Sites; D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000032:analysis*; D005038:Ethylnitrosourea / Q000031:analogs & derivatives / Q000494:pharmacology; D005374:Filtration; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013997:Time Factors
D003432:Cross-Linking Reagents; C017139:1-(2-chloroethyl)-1-nitrosourea; D004247:DNA; D005038:Ethylnitrosourea; D002945:Cisplatin