A polymerase chain reaction-based method to detect cisplatin adducts in specific genes.
Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.
Jennerwein
M M
MM
Department of Pharmacology, Dartmouth Medical School, Hanover, NH 03756.
Eastman
A
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002945:Cisplatin / Q000633:toxicity*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D004996:Ethidium; D002945:Cisplatin