Abstract IA20: Novel cell microarray technologies for studies of DNA damage and cell survival and applications for studies of microbe-induced DNA damage
| Autor: | Les Recio, Yang Su, Matthew R. Wilson, Prashant Rai, Carole Swartz, John Winters, Bevin P. Engelward, Vincent T. K. Chow, Leona D. Samson, Tze-Khee Chan, Emily P. Balskus, Jing Ge, Le P. Ngo |
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| Rok vydání: | 2020 |
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| Zdroj: | Cancer Prevention Research. 13:IA20-IA20 |
| ISSN: | 1940-6215 1940-6207 |
| Popis: | Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The comet assay is a commonly used approach for detecting DNA damage that is based on the underlying principle that damaged DNA migrates more readily than undamaged DNA when electrophoresed in agarose. We previously developed a higher-throughput version of the comet assay (Wood DK, PNAS 2010). For the “CometChip” assay, cells are loaded into an array of agarose microwells to create a cell microarray. The CometChip can be fully automated, making the assay more than 1000X faster, while improving reproducibility. While effective for detecting single-strand breaks, abasic sites, and alkali-sensitive sites, the alkaline comet assay is not effective for detection of carcinogenic bulky DNA lesions that do not impact DNA mobility and that require metabolic activation. To overcome this, we use DNA replication inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) shown to trap single-strand breaks that are formed during nucleotide excision repair, which normally removes bulky lesions. Thus, comet-undetectable bulky lesions are converted into comet-detectable single-strand breaks. Together with HepaRG™ cells that recapitulate in vivo metabolic capacity, we have thus created the “HepaCometChip,” which provides a more broadly effective approach for detection of DNA damage. In addition to genotoxicity testing, the microwell array has also been harnessed for use in a cell survival assay. For the “MicroColonyChip” (Ngo LP, Cell Reports 2019), cells are loaded into the agarose microwell array and allowed to grow to form microcolonies. Differences in cell survival can be detected by changes in the distribution of colony sizes, a novel metric for cell survival quantitation. The assay is as sensitive as the traditional “gold standard” colony-forming assay, yet it takes days instead of weeks to complete. Parallel studies of genotoxicity and cytotoxicity can thus be performed using a shared microwell platform. These tools have broad utility, including studies of microbe-induced DNA damage. Examples of S. pneumoniae and E. coli microbial product-induced double-strand breaks and interstrand crosslinks contribute to an emerging paradigm wherein pathogen-induced DNA damage has the potential to promote disease. Citation Format: Le P. Ngo, Prashant Rai, Matthew R. Wilson, Carole Swartz, John Winters, Yang Su, Jing Ge, Tze-Khee Chan, Emily P. Balskus, Vincent Chow, Les Recio, Leona D. Samson, Bevin P. Engelward. Novel cell microarray technologies for studies of DNA damage and cell survival and applications for studies of microbe-induced DNA damage [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr IA20. |
| Databáze: | OpenAIRE |
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