Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction.
We examined ultraviolet (UV) irradiation and cisplatin treatment damage formation and repair efficiency in the p53 tumor suppressor gene of various cultured cell lines and lymphocytes using a nonradioactive multiplex long quantitative polymerase chain reaction (QPCR) assay, which amplified a 7-kb fragment of the target gene and a 500-bp fragment of the template control to successfully increase the sensitivity and reliability of the assay. The multiplex long QPCR detected a lesion frequency of 0.63 lesions/10kb/10J/m(2) in the p53 gene of fibroblast cells. In addition, the multiplex long QPCR assay detected pronounced differences in the repair of UV damage in the p53 gene among repair-proficient CRL-1475 cells and repair-deficient XP-A and XP-C cells. The multiplex long QPCR assay was also evaluated as a sensitive assay for the detection of DNA damage induced by cisplatin. The data indicated that the lesion frequency in the p53 gene was 1.27-1.75 times higher in the H23 cisplatin-sensitive cell than in the H1435 cisplatin-resistant cell at the IC(70) dose. After 8-h and 24-h repair periods, only 13 and 75% of cisplatin-induced damage had been removed in the H23 cells, whereas these values were 92 and 100% in the H1435 cells. In addition, our data indicate that multiplex long QPCR is a sensitive method for validly estimating repair in freshly isolated lymphocytes. The results suggest that the current protocol of the multiplex long QPCR method can be used to assess the damage formation and repair efficiency of various agents at biologically relevant doses and to allow a more precise determination of gene-specific repair in disease susceptibility and drug resistance in epidemiological studies.
Wang
Yu-Chieh
YC
Department of Biology, National Taiwan Normal University, Taipei, Taiwan, ROC. t43017@cc.ntnu.edu.tw
Lee
Pei-Jung
PJ
Shih
Chuen-Ming
CM
Chen
Hsing-Yu
HY
Lee
Chin-Chu
CC
Chang
Yuan-Yen
YY
Hsu
Yu-Ting
YT
Liang
Ying-Ju
YJ
Wang
Li-Ya
LY
Han
Wen-Hua
WH
Wang
Yi-Ching
YC
D001345:Autoradiography; D002460:Cell Line; D002945:Cisplatin / Q000633:toxicity; D004249:DNA Damage / Q000235:genetics*; D053938:DNA Fragmentation / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000502:physiology; D016158:Genes, p53 / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D014466:Ultraviolet Rays / Q000009:adverse effects
D002945:Cisplatin