A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.
X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.
Forrester
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HB
Radiation Oncology Research Laboratory, University of California San Francisco, MCB 200, 1855 Folsom Street, San Francisco, California 94103-0806, USA.
Yeh
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RF
Dewey
W C
WC
D002877:Chromosomes, Human / Q000528:radiation effects*; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000235:genetics / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005544:Forecasting; D016259:Genes, myc / Q000528:radiation effects; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics
D004273:DNA, Neoplasm