Establishment of a Microplate-Formatted Cell-Based Immunoassay for Rapid Analysis of Nucleotide Excision Repair Ability in Human Primary Cells
Establishment of a Microplate-Formatted Cell-Based Immunoassay for Rapid Analysis of Nucleotide Excision Repair Ability in Human Primary Cells
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DOI：
10.1111/j.1751-1097.2012.01073.x
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发表时间：
2012-03-01
影响因子：
3.3
通讯作者：
Matsunaga, Tsukasa
中科院分区：
文献类型：
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作者：
Nishinaga, Mari;Kurata, Ryuichiro;Matsunaga, Tsukasa
紫外线、环丁烷嘧啶二聚体（CPD）和（6-4）光产物（6-4PP）诱导的DNA光损伤，在人细胞中可通过核苷酸切除修复（NER）来修复。已经开发了使用特异于光损伤的单克隆抗体的各种免疫测定，并广泛用于细胞NER活性的分析。在这项研究中，我们新开发了一种微孔板格式的细胞为基础的免疫测定，基于间接免疫荧光染色与病变特异性抗体结合红外成像系统。使用该测定，我们显示了来自新生儿和成人供体的各种成纤维细胞中CPD和6-4PP的修复动力学，没有年龄相关的差异。此外，表皮角质形成细胞和黑素细胞表现出相当的NER活性，和钙离子诱导的角质形成细胞的分化没有显着影响其NER活性。我们还评估了蛋白酶体抑制剂MG 132和组蛋白脱乙酰酶抑制剂丁酸钠对NER效率的影响。所有这些结果表明，新的检测方法是非常有用的快速和定量分析的NER活性在各种原代细胞有限的生长活性，并适用于筛选系统的药物影响NER效率。
DNA photolesions induced by UV, cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP), are repaired by nucleotide excision repair (NER) in human cells. Various immunoassays using monoclonal antibodies specific for the photolesions have been developed and widely used for the analysis of cellular NER activity. In this study, we have newly developed a microplate-formatted cell-based immunoassay, based on indirect immunofluorescence staining with lesion-specific antibodies combined with an infrared imaging system. Using this assay, we show the repair kinetics of CPD and 6-4PP in various fibroblasts from newborn and adult donors with no age-related difference. Furthermore, epidermal keratinocytes and melanocytes exhibit comparable NER activity, and calcium ion-induced differentiation of keratinocytes has no significant impacts on their NER activity. We also evaluated the effects of a proteasome inhibitor, MG132, and a histone deacetylase inhibitor, sodium butyrate, on NER efficiency using this assay. All these results suggest that the new assay is highly useful for the rapid and quantitative analysis of NER activity in various primary cells with limited growth activity and is applicable to a screening system for drugs affecting NER efficiency.