Abstract
BackgroundWe have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity.ResultsHDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency.ConclusionsHDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival.
Highlights
DNA double strand breaks (DSBs) are toxic lesions that represent a major threat to cell survival
We found that histone deacetylase inhibitor (HDACi), including trichostatin A (TSA), increased the protein level of ATAD5 {Figure 1a and [25]} and activated DNA damage responses (Figure 1b)
Transformed cells are usually highly susceptible to HDACi drugs such as TSA or suberoylanilide hydroxamic acid (SAHA) [31] which induce DNA damage (Figure 1b); we reasoned that DNA repair pathways such as homology dependent repair (HDR) and non-homologous end joining (NHEJ) are likely important in defining drug sensitivity in tumors
Summary
DNA double strand breaks (DSBs) are toxic lesions that represent a major threat to cell survival. The RAD51 strand exchange protein is a central player in HDR and mediates the process of synapse from a wild type allele {for review see [2]}. Other HDR proteins generally function as positive or negative regulators of RAD51. Negative regulators of HDR such as BLM and FANCM have evolved to prevent uncontrolled or unscheduled HDR [5], which could lead to genomic DNA rearrangements [2]. Mutations in BRCA2 and/or BRCA1 increase predisposition to breast and ovarian cancers [4], while mutations in BLM or FANCM are associated with syndromes showing elevated genomic instability and cancer susceptibility in general [2,10]. We used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity
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