Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells

In concurrent chemoradiotherapy, drugs are used to sensitize tumors to ionizing radiation. Although a spectrum of indications for simultaneous treatment with drugs and radiation has been defined, the molecular mechanisms underpinning tumor radiosensitization remain incompletely characterized for several such combinations. Here, we investigate the mechanisms of radiosensitization by the arabinoside nucleoside analogue 9-b-D-arabinofuranosyladenine (araA) placing particular emphasis on the repair of DNA double-strand breaks (DSB), and compare the results to those obtained with fludarabine

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“…The reporter constructs integrated in U2OS cells have been described (28,29). DRaa40 cells were grown in DMEM.…”

Section: Methodsmentioning

confidence: 99%

“…As the spectrum of errors reported by the EJ5-GFP substrate is limited to a single event, we introduced an alternative system designed to report a wide spectrum of deletions and other mutagenic events (29). In this two component system (Fig.…”

Section: Treatment With Araa Enhances Utilization Of Mutagenic Dsb Rementioning

confidence: 99%

Inhibition of Homologous Recombination and Promotion of Mutagenic Repair of DNA Double-Strand Breaks Underpins Arabinoside–Nucleoside Analogue Radiosensitization

Magin

Papaioannou

Saha

et al. 2015

Molecular Cancer Therapeutics

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“…Collectively, the non-canonical NHEJ repair processes share a common theme of higher rates of insertions, deletions, and microhomology usage. As such, we have termed this pathway mutagenic NHEJ (mNHEJ) repair previously, in order to distinguish cNHEJ repair versus bNHEJ, aNHEJ, MMEJ, which often are used interchangeably but sometimes distinctly (27). However, MMEJ repair specifically may represent a subset of mNHEJ in which flanking sequence microhomology is commonly (if not exclusively) utilized.…”

Section: Introductionmentioning

confidence: 99%

“…The DR-GFP assay is a commonly employed HR assay, which uses the I-SceI endonuclease to induce a site-specific DSB in a cell (33). We recently developed a novel mNHEJ repair assay, termed End Joining-Red Fluorescent Protein (EJ-RFP), which can be combined with DR-GFP to measure both DSB repair pathways simultaneously (27). We integrated this system into a number of cells, including U2OS DR-GFP cells (referred to as U2OS EJ-DR cells).…”

Section: Introductionmentioning

confidence: 99%

Identification of Novel Radiosensitizers in a High-Throughput, Cell-Based Screen for DSB Repair Inhibitors

Goglia

Delsite

Luz

et al. 2015

Molecular Cancer Therapeutics

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Most cancer therapies involve a component of treatment which inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radio-sensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules which selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits which potently inhibit DSB repair, and we have validated their functional activity in comprehensive panel of orthogonal secondary assays. A selection of these inhibitors were found to radiosensitize cancer cell lines in vitro, which suggests they may be useful as novel chemo- and radio-sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker, mibefradil dihydrochloride, which demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a Phase I clinical trial testing mibefradil as glioma radiosensitizer.

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“…As described by Bindra et al [45] , non-homologous end-joining (NHEJ) and HRR comprise the two major pathways by which double-strand breaks (DSBs) are repaired in cells. NHEJ processes and re-ligates the exposed DNA termini of DSBs without the use of significant homology, whereas HRR uses homologous DNA sequences as a template for repair.…”

Section: Mutations Are Frequently Caused By Error-prone Repair Of Doumentioning

confidence: 99%

Epigenetic reduction of DNA repair in progression to gastrointestinal cancer

Bernstein

2015

WJGO

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Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy.

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Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells

Cited by 75 publications

References 67 publications

Inhibition of Homologous Recombination and Promotion of Mutagenic Repair of DNA Double-Strand Breaks Underpins Arabinoside–Nucleoside Analogue Radiosensitization

Inhibition of Homologous Recombination and Promotion of Mutagenic Repair of DNA Double-Strand Breaks Underpins Arabinoside–Nucleoside Analogue Radiosensitization

Identification of Novel Radiosensitizers in a High-Throughput, Cell-Based Screen for DSB Repair Inhibitors

Epigenetic reduction of DNA repair in progression to gastrointestinal cancer

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