A polymerase chain reaction-based method to detect cisplatin adducts in specific genes

Abstract

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.

DNA damage by anti-cancer agents resolved at the nucleotide level of a single copy gene: evidence for a novel binding site for cisplatin in cells

“…It is likely that all cisplatin adducts formed are detected by sslig-PCR. A quantitative PCR assay has demonstrated that taq polymerase is completely blocked by one adduct per DNA strand (13), and Comess et al (20) have shown that, in artificial systems, adduct bypass by taq polymerase is at most 3 % for GG crosslinks and 19% for AG crosslinks. It can be seen that adducts on pairs of guanines are represented by two bands on the autoradiograph whereas only one might be expected.…”

“…The lack of techniques to measure precise DNA binding within intact cells has also meant that the sequence selectivity of repair of individual lesions is a largely unexplored area. Southern blotting and PCR based methods currently available for the measurement of DNA repair in the intact cell can do so at the level of the gene (10-20 kb) (9,10) and regions within the gene (500-2000 bp) (11)(12)(13) respectively, but cannot give information at the level of the individual base.…”

ATF3 and p15PAF are novel gatekeepers of genomic integrity upon UV stress

“…UV adduct formation and repair were analyzed by a PCR-based DNA damage assay (PCR stop assay). 14 HaCaT cells (10 6 cells in 60 mm dishes) were either left untreated or UVC-irradiated (5 J/m 2 ). After 2 and 24 h, a 2-kb region of HPRT gene (primer available upon request) was amplified by PCR (20 cycles) from genomic DNA.…”

The Jun Kinase/Stress-activated Protein Kinase Pathway Functions to Regulate DNA Repair and Inhibition of the Pathway Sensitizes Tumor Cells to Cisplatin

“…Cisplatin Activates and dnJun Inhibits DNA Repair-We assessed the extent of genomic DNA damage and repair following cisplatin treatment using a modified PCR assay (25). For this assay, it has been shown that the degree of inhibition of PCR-catalyzed amplification of DNA purified from cisplatintreated cells is a direct measure of the amount of DNA-cisplatin adduct formation as measured by atomic absorption (25).…”

“…For this assay, it has been shown that the degree of inhibition of PCR-catalyzed amplification of DNA purified from cisplatintreated cells is a direct measure of the amount of DNA-cisplatin adduct formation as measured by atomic absorption (25). Thus, this assay provides a direct assessment of the extent of cisplatin-induced DNA damage.…”