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High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death
Citation:
SIMMONS, S., J. T. Fox, S. Sakamuru, R. Huang, N. Teneva, M. Xia, R. B. Tice, C. P. Austin, AND K. Myung. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. National Academy of Sciences, WASHINGTON, DC, 109(14):5423-5428, (2012).
Impact/Purpose:
This study unveils a new cell-based HTS assay for genotoxicity using a novel luciferase reporter strategy to exploit protein stabilzation as an endpoint for pathway activation. This assay was used to identify known and novel genotoxicants from a large chemical library.
Description:
Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can monitor qenotoxicity. Using this assay to screen chemical libraries, we identified several antioxidants including resveratrol, genistein and baicalein as genotoxic compounds. In contrast to the previously known beneficial effects of antioxidants, we found that these compounds induced DNA damage in rapidly dividing cells and resulted in cell death. Importantly, resveratrol, genistein and baicalein did not cause any mutagenic effects at the chromosomal or nucleotide levels in comparison to the major side effects of conventional anti-cancer drugs. Therefore, we propose that resveratrol, genistein and baicalein are attractive candidatesfor improved chemotherapeutic agents. One major distinctive characteristic of cancer cells is their persistent cell division that requires duplication of their genome by DNA replication. This feature is often exploited in the development of chemotherapeutic drugs since the inhibition of DNA replication could specifically kill cancer cells. The most common mechanisms of replication inhibition used in the clinical setting involve the induction of DNA damage by radiation or genotoxic chemicals. DNA lesions resulting from exposure to these agents stall DNA replication, collapse replication forks, and produce DNA double-strand breaks (DSBs), resulting in cancer cell death. However, such genomic insults could also induce mutagenesis or chromosomal rearrangements that ultimately might potentiate the development of secondary tumors. Thus, cancer treatment will greatly benefit from the identification of new genotoxic agents that kill rapidly dividing cells with minimal mutagenetic side effects. ATAD5 is the homolog of yeast Enhanced Level of Genome Instability Gene 1 (ELG1), which makes a heteropentameric alternative replication factor C complex and suppresses genomic instability (1-6). ATAD5 plays a key role in the translesion synthesis (TLS). pathway where multiple TLS polymerases are used to bypass DNA lesions that stall or collapse GNA replications forks (7,8).