Ahn Byungchan, Kang Daehee, Kim Hyangsook, Wei Qingyi
Department of Life Sciences, University of Ulsan, Ulsan 680-749, Korea.
Mol Cells. 2004 Oct 31;18(2):249-55.
DNA repair capacity in a cell could be detected by a host-cell reactivation assay (HCR). Since relation between DNA repair and genetic susceptibility to cancer remains unclear, it is necessary to identify DNA repair defects in human cancer cells. To assess DNA repair for breast cancer susceptibility, we developed a modified HCR assay using a plasmid containing a firefly luciferase gene damaged by mitomycin C (MMC), which forms interstrand cross-link (ICL) adducts. In particular, interstrand cross-link is thought to induce strand breaks being repaired by homologous recombination. The MMC-ICLs were verified by electrophoresis. Damaged plasmids were transfected into apparently normal human lymphocytes and NER-deficient XP cell lines and the DNA repair capacity of the cells were measured by quantifying the activity of the firefly luciferase. MMC lesion was repaired as much as UV adducts in normal lymphocytes and the XPC cells. However, the XPA cells have a lower repair capacity for MMC lesion than the XPC cell, indicating that the XPA protein may be involved in initial damage recognition of MMC-ICL adducts. Since several repair pathways including NER and recombination participate in MMC-ICL removal, this host cell reactivation assay using MMC-ICLs can be used in exploring DNA repair defects in human cancer cells.
细胞中的DNA修复能力可通过宿主细胞再激活试验（HCR）来检测。由于DNA修复与癌症遗传易感性之间的关系尚不清楚，因此有必要在人类癌细胞中鉴定DNA修复缺陷。为了评估乳腺癌易感性的DNA修复情况，我们开发了一种改良的HCR试验，使用一个含有被丝裂霉素C（MMC）损伤的萤火虫荧光素酶基因的质粒，MMC会形成链间交联（ICL）加合物。特别是，链间交联被认为会诱导通过同源重组修复的链断裂。通过电泳验证了MMC-ICL。将受损质粒转染到外观正常的人类淋巴细胞和NER缺陷的XP细胞系中，并通过定量萤火虫荧光素酶的活性来测量细胞的DNA修复能力。在正常淋巴细胞和XPC细胞中，MMC损伤的修复程度与紫外线加合物相同。然而，XPA细胞对MMC损伤的修复能力低于XPC细胞，这表明XPA蛋白可能参与MMC-ICL加合物的初始损伤识别。由于包括NER和重组在内的几种修复途径参与了MMC-ICL的去除，这种使用MMC-ICL的宿主细胞再激活试验可用于探索人类癌细胞中的DNA修复缺陷。
