Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction

Yu-Chieh Wang, Pei Jung Lee, Chuen Ming Shih, Hsing Yu Chen, Chin Chu Lee, Yuan Yen Chang, Yu Ting Hsu, Ying Ju Liang, Li Ya Wang, Wen Hua Han, Yi Ching Wang

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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/10 kb/10 J/m2 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 IC70 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.

Original languageEnglish
Pages (from-to)206-215
Number of pages10
JournalAnalytical Biochemistry
Volume319
Issue number2
DOIs
StatePublished - 15 Aug 2003

Fingerprint

Lymphocytes
Polymerase chain reaction
p53 Genes
Repair
Blood
Genes
Cells
Cisplatin
Cell Line
Polymerase Chain Reaction
Assays
Disease Resistance
Disease Susceptibility
Tumor Suppressor Genes
Drug Resistance
Fibroblasts
DNA Damage
Epidemiologic Studies
Cultured Cells
Tumors

Keywords

  • Cisplatin
  • DNA repair
  • Quantitative PCR
  • UV
  • p53 gene

Cite this

Wang, Yu-Chieh ; Lee, Pei Jung ; Shih, Chuen Ming ; Chen, Hsing Yu ; Lee, Chin Chu ; Chang, Yuan Yen ; Hsu, Yu Ting ; Liang, Ying Ju ; Wang, Li Ya ; Han, Wen Hua ; Wang, Yi Ching. / Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction. In: Analytical Biochemistry. 2003 ; Vol. 319, No. 2. pp. 206-215.
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abstract = "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/10 kb/10 J/m2 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 IC70 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.",
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Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction. / Wang, Yu-Chieh; Lee, Pei Jung; Shih, Chuen Ming; Chen, Hsing Yu; Lee, Chin Chu; Chang, Yuan Yen; Hsu, Yu Ting; Liang, Ying Ju; Wang, Li Ya; Han, Wen Hua; Wang, Yi Ching.

In: Analytical Biochemistry, Vol. 319, No. 2, 15.08.2003, p. 206-215.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction

AU - Wang, Yu-Chieh

AU - Lee, Pei Jung

AU - Shih, Chuen Ming

AU - Chen, Hsing Yu

AU - Lee, Chin Chu

AU - Chang, Yuan Yen

AU - Hsu, Yu Ting

AU - Liang, Ying Ju

AU - Wang, Li Ya

AU - Han, Wen Hua

AU - Wang, Yi Ching

PY - 2003/8/15

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N2 - 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/10 kb/10 J/m2 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 IC70 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.

AB - 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/10 kb/10 J/m2 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 IC70 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.

KW - Cisplatin

KW - DNA repair

KW - Quantitative PCR

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KW - p53 gene

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