Establishment of a microplate-formatted cell-based immunoassay for rapid analysis of nucleotide excision repair ability in human primary cells.
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.
Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan.
Mari
M
Nishinaga
Ryuichiro
R
Kurata
Kazuyoshi
K
Onishi
Kenichi
K
Kuriyama
Mitsuo
M
Wakasugi
Tsukasa
T
Matsunaga
D000328:Adult; D002087:Butyrates / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007231:Infant, Newborn; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D007976:Leupeptins / Q000494:pharmacology; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D061251:Primary Cell Culture; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011740:Pyrimidine Dimers / Q000494:pharmacology; D013055:Spectrophotometry, Infrared; D014466:Ultraviolet Rays
D002087:Butyrates; D007976:Leupeptins; D061988:Proteasome Inhibitors; D011740:Pyrimidine Dimers; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D002118:Calcium