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Immunodeficiency patients with DNA repair defects exhibit radiosensitivity and proneness to leukemia/lymphoma formation. Though progress has been made in identifying the underlying mutations, in most patients the genetic basis is unknown. Two de novo mutated candidate genes, MCM3AP encoding germinal center-associated nuclear protein (GANP) and POMP encoding proteasome maturation protein (POMP), were identified by whole-exome sequencing (WES) and confirmed by Sanger sequencing in a child with complex phenotype displaying immunodeficiency, genomic instability, skin changes, and myelodysplasia. GANP was previously described to promote B-cell maturation by nuclear targeting of activation-induced cytidine deaminase (AID) and to control AID-dependent hyperrecombination. POMP is required for 20S proteasome assembly and, thus, for efficient NF-κB signaling. Patient-derived cells were characterized by impaired homologous recombination, moderate radio- and cross-linker sensitivity associated with accumulation of damage, impaired DNA damage-induced NF-κB signaling, and reduced nuclear AID levels. Complementation by wild-type (WT)-GANP normalized DNA repair and WT-POMP rescued defective NF-κB signaling. In conclusion, we identified for the first time mutations in MCM3AP and POMP in an immunodeficiency patient. These mutations lead to cooperative effects on DNA recombination and damage signaling. Digenic/polygenic mutations may constitute a novel genetic basis in immunodeficiency patients with DNA repair defects.

Hematopoietic stem and progenitor cells (HSPC), that is, the cell population giving rise not only to all mature hematopoietic lineages but also the presumed target for leukemic transformation, can transmit (adverse) genetic events, such as are acquired from chemotherapy or ionizing radiation. Data on the repair of DNA double-strand-breaks (DSB) and its accuracy in HSPC are scarce, in part contradictory, and mostly obtained in murine models. We explored the activity, quality and molecular components of DSB repair in human HSPC as compared with mature peripheral blood lymphocytes (PBL). To consider chemotherapy/radiation-induced compensatory proliferation, we established cycling HSPC cultures. Comparison of pathway-specific repair activities using reporter systems revealed that HSPC were severely compromised in non-homologous end joining and homologous recombination but not microhomology-mediated end joining. We observed a more pronounced radiation-induced accumulation of nuclear 53BP1 in HSPC relative to PBL, despite evidence for comparable DSB formation from cytogenetic analysis and γH2AX signal quantification, supporting differential pathway usage. Functional screening excluded a major influence of phosphatidylinositol-3-OH-kinase (ATM/ATR/DNA-PK)- and p53-signaling as well as chromatin remodeling. We identified diminished NF-κB signaling as the molecular component underlying the observed differences between HSPC and PBL, limiting the expression of DSB repair genes and bearing the risk of an inaccurate repair.

NF-{kappa}B is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-{kappa}B can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-{kappa}B in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-{kappa}B targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-{kappa}B required the targets ATM and BRCA2. Additionally, we provide evidence that NF-{kappa}B interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-{kappa}B activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-{kappa}B-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-{kappa}B-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-{kappa}B activation.

Ataxia telangiectasia mutated (ATM) has multiple functions in homologous recombination (HR) and nonhomologous end joining (NHEJ), which lead to conflicting data regarding its DNA double-strand break-repair (DSBR) functions in previous studies. To explore the effect of clinically relevant ATM mutations, we characterized DSBR between mutated EGFP genes and ATM kinase signaling in 9 lymphoblastoid cell lines (LCLs) derived from patients with ataxia telangiectasia (AT) with defined vs. 3 control LCLs without ATM mutations. Our study revealed that the DSBR phenotype in AT cells is not uniform but appears to depend on the mutation, causing up to 32-fold increased or up to 3-fold decreased activities in particular pathways. Comparison with a further 10 LCLs mutated in downstream factors (BRCA1, BRCA2, Nibrin, Rad50, and Chk2) showed that the most diametrically opposed DSBR patterns in AT cells phenocopied NBN/RAD50 or BRCA1 mutations. Notably, reexpressing wild-type ATM reversed these defects by 2.3- to 3.5-fold. Our data suggest that ATM stimulates repair proteins such as Nibrin, which execute HR, single-strand annealing (SSA), and NHEJ. Concomitantly, ATM minimizes error-prone repair (SSA and NHEJ) through activation of surveillance factors such as BRCA1. Since the outcome of the individual defect can be diametrically opposed, distinguishing repair patterns in patients with ATM mutations may also be relevant regarding therapeutic responses.

Microcystins, which are hepatotoxins produced by cyanobacteria, have been reported to be potent tumour promoters, and there is an indication that they can also act as tumour initiators. They thus constitute a potential threat to human and animal health, at concentrations that do not cause acute hepatotoxic effects. The main target organ of microcystin toxicity is the liver; however, several studies have shown that other organs and tissues may also be affected. We have investigated the effect of non-cytotoxic concentrations of microcystin-LR (MCLR) on the generation of intracellular reactive oxygen species (ROS) and on DNA damage in human colon adenocarcinoma CaCo-2, human astrocytoma IPDDC-A2 and human B-lymphoblastoid NCNC cell lines. The viability of CaCo-2 cells exposed to 10 microg/MCLR for 24 and 48 h was reduced by about 40%, while that of NCNC and IPDDC-2A cells was not affected. Intracellular ROS production was increased in CaCo-2 and IPDDC-2A, but not NCNC, cells. Using the comet assay, it was shown that MCLR, at non-cytotoxic concentrations, induced a time and dose dependent increase of DNA damage in CaCo-2 cells, but not significantly in IPDDC-2A and NCNC cells. Thus, CaCo-2 cells were the most sensitive. Their sensitivity is comparable to that observed in our previous study with human hepatoma HepG2 cells. These results indicate that, in addition to liver cells, colon cells should also be considered as a target for microcystin toxicity, and that exposure to low doses of microcystins may affect intestinal tissue.

Immunodeficiency patients with DNA repair defects exhibit radiosensitivity and proneness to leukemia/lymphoma formation. Though progress has been made in identifying the underlying mutations, in most patients the genetic basis is unknown. Two de novo mutated candidate genes, MCM3AP encoding germinal center-associated nuclear protein (GANP) and POMP encoding proteasome maturation protein (POMP), were identified by whole-exome sequencing (WES) and confirmed by Sanger sequencing in a child with complex phenotype displaying immunodeficiency, genomic instability, skin changes, and myelodysplasia. GANP was previously described to promote B-cell maturation by nuclear targeting of activation-induced cytidine deaminase (AID) and to control AID-dependent hyperrecombination. POMP is required for 20S proteasome assembly and, thus, for efficient NF-κB signaling. Patient-derived cells were characterized by impaired homologous recombination, moderate radio- and cross-linker sensitivity associated with accumulation of damage, impaired DNA damage-induced NF-κB signaling, and reduced nuclear AID levels. Complementation by wild-type (WT)-GANP normalized DNA repair and WT-POMP rescued defective NF-κB signaling. In conclusion, we identified for the first time mutations in MCM3AP and POMP in an immunodeficiency patient. These mutations lead to cooperative effects on DNA recombination and damage signaling. Digenic/polygenic mutations may constitute a novel genetic basis in immunodeficiency patients with DNA repair defects.

NF-kappaB is activated by DNA-damaging anticancer drugs as part of the cellular stress response. However, the consequences of drug-induced NF-kappaB activation are still only partly understood. To investigate the impact of NF-kappaB on the cell's response to DNA damage, we engineered glioblastoma cells that stably express mutant IkappaBalpha superrepressor (IkappaBalpha-SR) to block NF-kappaB activation. Here, we identify a novel pro-apoptotic function of NF-kappaB in the DNA damage response in glioblastoma cells. Chemotherapeutic drugs that intercalate into DNA and inhibit topoisomerase II such as Doxorubicin, Daunorubicin and Mitoxantrone stimulate NF-kappaB DNA binding and transcriptional activity prior to induction of cell death. Importantly, specific inhibition of drug-induced NF-kappaB activation by IkappaBalpha-SR or RNA interference against p65 significantly reduces apoptosis upon treatment with Doxorubicin, Daunorubicin or Mitoxantrone. NF-kappaB exerts this pro-apoptotic function especially after pulse drug exposure as compared to continuous treatment indicating that the contribution of NF-kappaB becomes relevant during the recovery phase following the initial DNA damage. Mechanistic studies show that NF-kappaB inhibition does not alter Doxorubicin uptake and efflux or cell cycle alterations. Genetic silencing of p53 by RNA interference reveals that NF-kappaB promotes drug-induced apoptosis in a p53-independent manner. Intriguingly, drug-mediated NF-kappaB activation results in a significant increase in DNA damage prior to the induction of apoptosis. By demonstrating that NF-kappaB promotes DNA damage formation and apoptosis upon pulse treatment with DNA intercalators, our findings provide novel insights into the control of the DNA damage response by NF-kappaB in glioblastoma.