Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts
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...
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| Format: | Article |
| Language: | English |
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Sciendo
2015-09-01
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| Series: | Acta Pharmaceutica |
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| Online Access: | https://doi.org/10.1515/acph-2015-0025 |
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| _version_ | 1832572492478152704 |
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| author | Bae Seunghee An In-Sook An Sungkwan |
| author_facet | Bae Seunghee An In-Sook An Sungkwan |
| author_sort | Bae Seunghee |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-85addfdc049343d5b7acc7971b1bfc5d |
| institution | Kabale University |
| issn | 1846-9558 |
| language | English |
| publishDate | 2015-09-01 |
| publisher | Sciendo |
| record_format | Article |
| series | Acta Pharmaceutica |
| spelling | doaj-art-85addfdc049343d5b7acc7971b1bfc5d2025-02-02T09:57:48ZengSciendoActa Pharmaceutica1846-95582015-09-0165333134110.1515/acph-2015-0025acph-2015-0025Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblastsBae Seunghee0An In-Sook1An Sungkwan2Korea Institute for Skin and Clinical Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of KoreaKorea Institute for Skin and Clinical Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of KoreaKorea Institute for Skin and Clinical Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of KoreaUltraviolet (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.https://doi.org/10.1515/acph-2015-0025human dermal fibroblastsdna damagehigh-throughput screeningaging |
| spellingShingle | Bae Seunghee An In-Sook An Sungkwan Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts Acta Pharmaceutica human dermal fibroblasts dna damage high-throughput screening aging |
| title | Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts |
| title_full | Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts |
| title_fullStr | Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts |
| title_full_unstemmed | Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts |
| title_short | Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts |
| title_sort | development of a high throughput screening system for identification of novel reagents regulating dna damage in human dermal fibroblasts |
| topic | human dermal fibroblasts dna damage high-throughput screening aging |
| url | https://doi.org/10.1515/acph-2015-0025 |
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