title: Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.
abstract: Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.
authors.forename: Seunghee
authors.initials: S
authors.lastname: Bae
authors.forename: In-Sook
authors.initials: IS
authors.lastname: An
authors.forename: Sungkwan
authors.initials: S
authors.lastname: An
mesh_terms: D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects*; D005347:Fibroblasts / Q000187:drug effects*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry* / Q000494:pharmacology*; D012867:Skin / Q000187:drug effects; D015595:Skin Aging / Q000187:drug effects; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D007202:Indicators and Reagents