Quantitation of gene-specific DNA damage by competitive PCR.
A sensitive assay for quantitating DNA damage within individual genes would be a valuable tool for identifying the molecular mechanisms of disease and the sites of action of various carcinogens and anticancer drugs. This report describes a competitive PCR assay that was used to quantitate DNA damage induced by anticancer drugs within a 683-bp region of the c-myc gene in human CEM leukemia cells. Absolute quantitation of gene-specific DNA damage (attomoles or molecules of damaged DNA sequences) was achieved by coamplification of a homologous internal standard that has the same primer binding sites and PCR amplification efficiency as c-myc. The variability (standard error) associated with four separate determinations of the amount of c-myc sequence in 300 ng of DNA from untreated cells (6.80 +/- 0.05 SE amol) was less than 1% of the mean. The assay was capable of quantitating direct DNA damage that was induced by therapeutic concentrations of VM-26 and cisplatin prior to the onset of cellular apoptosis or necrosis. Both VM-26 (1-10 microM) and cisplatin (25-100 microM) induced a dose-dependent decrease in the amount of intact c-myc sequence. This assay should be readily adaptable to current real-time PCR protocols.
Fernando
Lawrence P
LP
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA.
Kurian
Philip J
PJ
Fidan
Melihat
M
Fernandes
Daniel J
DJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D016259:Genes, myc; D006801:Humans; D009336:Necrosis; D016133:Polymerase Chain Reaction / Q000379:methods*; D013713:Teniposide / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D013713:Teniposide; D002945:Cisplatin