Immortalization and characterization of Nijmegen Breakage syndrome fibroblasts.

Nijmegen Breakage Syndrome (NBS) is a very rare autosomal recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency and a high incidence of malignancies. Cells from NBS patients are hypersensitive to ionizing radiation (IR) and display radioresistant DNA synthesis (RDS). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 encoding a protein called nibrin. This protein is a component of the hMre11/hRad50 protein complex, suggesting a defect in DNA double-strand break (DSB) repair and/or cell cycle checkpoint function in NBS cells. We established SV40 transformed, immortal NBS fibroblasts, from primary cells derived from a Polish patient, carrying the common founder mutation 657del5. Immortalized NBS cells, like primary cells, are X-ray sensitive (2-fold) and display RDS following IR. They show an increased sensitivity to bleomycin (3.5-fold), etoposide (2.5-fold), camptothecin (3-fold) and mitomycin C (1.5-fold), but normal sensitivity towards UV-C. Despite the clear hypersensitivity towards DSB-inducing agents, the overall rates of DSB-rejoining in NBS cells as measured by pulsed field gel electrophoresis were found to be very similar to those of wild type cells. This indicates that the X-ray sensitivity of NBS cells is not directly caused by an overt defect in DSB repair.     

Genetic complementation analysis of ataxia telangiectasia and Nijmegen breakage syndrome: a survey of 50 patients

Cultured cells from patients with ataxia telangiectasia (AT) or Nijmegen breakage syndrome (NBS) are hypersensitive to ionizing radiation. After radiation exposure, the rate of DNA replication is inhibited to a lesser extent than in normal cells, whereas the frequency of chromosomal aberrations is enhanced. Both of these features have been used in genetic complementation studies on a limited series of patients. Here we report the results of extended complementation studies on fibroblast strains from 50 patients from widely different origins, using the radioresistant DNA replication characteristic as a marker. Six different genetic complementation groups were identified. Four of these, called AB, C, D, and E (of which AB is the largest), represent patients with clinical signs of AT. Patients having NBS fall into two groups, V1 and V2. An individual with clinical symptoms of both AT and NBS was found in group V2, indicating that the two disorders are closely related. In AT, any group-specific patterns with respect to clinical characteristics or ethnic origin were not apparent. In addition to the radiosensitive ATs, a separate category of patients exists, characterized by a relatively mild clinical course and weak radiosensitivity. It is concluded that a defect in one of at least six different genes may underlie inherited radiosensitivity in humans. To facilitate research on defined defects, a complete list of genetically characterized fibroblast strains is presented.     

Overproduction of topoisomerase II in an ataxia telangiectasia fibroblast cell line: comparison with a topoisomerase II-overproducing hamster cell mutant.

Ataxia telangiectasia (AT) cell lines are characterised by their hypersensitivity to ionizing radiation and bleomycin, and their failure to inhibit DNA synthesis after DNA damage. A recent report [Singh et al. (1988) Nucl. Acids Res. 16, 3919-3929] indicated that a reduction in topoisomerase II (topo II) activity was a feature of AT lymphoblast cell lines. We have studied the possible role of DNA topoisomerases in determining the phenotype of an AT fibroblast cell line. AT5BIVA cells are sensitive to the topo II inhibitors etoposide (VP16) and amsacrine (m-AMSA), compared to normal human fibroblasts (MRC5-V1 and VA13). AT5BIVA cells express a 3-fold higher level of topo II protein than MRC5-V1 cells, and 6-fold higher than VA13. This is reflected in elevated topo II activity in AT5BIVA cells. Untransformed AT5BI cells also show elevated topo II activity compared to untransformed normal cells. The extent of overproduction of topo II in AT5BIVA cells is comparable with that seen in a mutant Chinese hamster cell line, ADR-1, which is similarly hypersensitive to both bleomycin and topo II inhibitors. However, ADR-1 cells show neither hypersensitivity to ionizing radiation nor abnormal inhibition of DNA synthesis following DNA damage. Topo II overproduction per se does not appear sufficient to generate an "AT-like" phenotype. AT5BIVA cells express a reduced level of topoisomerase I (topo I) and are hypersensitive to the topo I inhibitor, camptothecin. ADR-1 cells express a normal level of topo I, indicating that a reduction in the level of topo I is not the inevitable consequence of an elevation in topo II.     

NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres

Nijmegen breakage syndrome, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. The NBS1 gene product, p95 (NBS1 or nibrin) forms a complex with Rad50 and Mre11. Cells deficient in the formation of this complex are defective in DNA double-strand break repair, cell cycle checkpoint control, and telomere length maintenance. How the NBS1 complex is involved in telomere length maintenance remains unclear. Here we show that the C-terminal region of NBS1 interacts directly with a telomere repeat binding factor, TRF1, by both yeast two-hybrid and in vivo DNA-coimmunoprecipitation assays. NBS1 and Mre11 colocalize with TRF1 at promyelocytic leukemia (PML) nuclear bodies in immortalized telomerase-negative cell lines, but rarely in telomerase-positive cell lines. The translocation of NBS1 to PML bodies occurs specifically during late S to G(2) phases of the cell cycle and coincides with active DNA synthesis in these NBS1-containing PML bodies. These results suggest that NBS1 may be involved in alternative lengthening of telomeres in telomerase-negative immortalized cells.     

Interaction of FANCD2 and NBS1 in the DNA damage response

Fanconi anaemia (FA) and Nijmegen breakage syndrome (NBS) are autosomal recessive chromosome instability syndromes with distinct clinical phenotypes. Cells from individuals affected with FA are hypersensitive to mitomycin C (MMC), and cells from those with NBS are hypersensitive to ionizing radiation. Here we report that both NBS cell lines and individuals with NBS are hypersensitive to MMC, indicating that there may be functional linkage between FA and NBS. In wild-type cells, MMC activates the colocalization of the FA subtype D2 protein (FANCD2) and NBS1 protein in subnuclear foci. Ionizing radiation activates the ataxia telangiectasia kinase (ATM)-dependent and NBS1-dependent phosphorylation of FANCD2, resulting in an S-phase checkpoint. NBS1 and FANCD2 therefore cooperate in two distinct cellular functions, one involved in the DNA crosslink response and one involved in the S-phase checkpoint response.     

NBS1 prevents chromatid-type aberrations through ATM-dependent interactions with SMC1

Antoccia, A., Sakamoto, S., Matsuura, S., Tauchi, H. and Komatsu, K., NBS1 Prevents Chromatid-Type Aberrations through ATM-Dependent Interactions with SMC1. Radiat. Res. 170, 345-352 (2008).Nijmegen breakage syndrome shares several common cellular features with ataxia telangiectasia, including chromosomal instability and aberrant S- and G(2)-phase checkpoint regulation. We show here that after irradiation, NBS1 interacts physically with both BRCA1 and SMC1, a component of the cohesin complex, and that their interactions are completely abolished in AT cells. It is noted that BRCA1 is required for the interaction of NBS1 with SMC1, whereas the reverse is not the case, since BRCA1 is able to bind to NBS1 in the absence of an NBS1/SMC1 interaction as observed in MRE11- or RAD50-deficient cells. This indicates that ATM and BRCA1 are upstream of the NBS1/SMC1 interaction. Furthermore, the interaction of NBS1 with SMC1 requires both conserved domains of NBS in the N-terminus and the C-terminus, since they are indispensable for binding of NBS1 to BRCA1 and to MRE11/ATM, respectively. The interaction of NBS1 with SMC1 and the resulting phosphorylation are compromised in the clones lacking either the N- or C-terminus of NBS1, and as a consequence, chromatid-type aberrations are enhanced after irradiation. Our results reveal that ATM plays a fundamental role in promoting the radiation-induced interaction of NBS1 with SMC1 in the presence of BRCA1, leading to the maintenance of chromosomal integrity.     

The NBS1-ATM Connection Revisited

Nijmegen Breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and increased predisposition to the development of malignancy.^1,2 Due to the overlap of clinical and cellular features of patients with ataxia telangiectasia (AT), NBS was described as an AT variant syndrome until the underlying gene product mutation was identified.^3-5 Cells from both AT and NBS patients show increased sensitivity to ionizing radiation (IR), genomic instability and cell cycle checkpoint defects following DNA damage,^6,7 suggesting that both gene products participate in the same DNA damage response pathway. Here we highlight recent developments and refinements in our understanding of the interplay between NBS1 and ATM in vivo.     

Expression of full-length NBS1 protein restores normal radiation responses in cells from Nijmegen breakage syndrome patients

Cells from Nijmegen breakage syndrome (NBS) display multiple phenotypes, such as chromosomal instability, hypersensitivity to cell killing from ionizing radiation, and possibly abnormal cell cycle checkpoints. NBS1, a gene mutated in NBS patients, appears to encode a possible repair protein, which could form the foci of a sensor-like molecular complex capable of detecting DNA double strand breaks, however, it has no kinase domain for signaling DNA damage. Here, we report that the stable expression of NBS1 cDNA in NBS cells after transfection results in the complete restoration of foci formation in the nucleus, and in normal cell survival after irradiation. The prolonged G2 block observed after irradiation was also abolished by expression of NBS1, providing additional confirmation that the G2 checkpoint is abrogated in NBS cells. These results suggest that a defective NBS1 protein could be the sole cause of the NBS phenotype, and that NBS1 likely interacts with another protein(s) to produce the entire range of NBS phenotypic expression.     

AT cells are not radiosensitive for simple chromosomal exchanges at low dose

Cells deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome) show increased yields of both simple and complex chromosomal aberrations after high doses (>0.5Gy) of ionizing radiation (X-rays or γ-rays), however less is known on how these cells respond at low dose. Previously we had shown that the increased chromosome aberrations in ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex exchanges. The linear dose-response term for simple exchanges was significantly higher in NBS cells compared to wild type cells, but not for AT cells. However, AT cells have a high background level of exchanges compared to wild type or NBS cells that confounds the understanding of low dose responses. To understand the sensitivity differences for high to low doses, chromosomal aberration analysis was first performed at low dose-rates (0.5Gy/d), and results provided further evidence for the lack of sensitivity for exchanges in AT cells below doses of 1Gy. Normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, showed increased numbers of exchanges at a dose of 1Gy and higher, but were similar to wild type cells at 0.5Gy or below. These results were confirmed using siRNA knockdown of ATM. The present study provides evidence that the increased radiation sensitivity of AT cells for chromosomal exchanges found at high dose does not occur at low dose.     

Association of ionizing radiation-induced foci of NBS1 with chromosomal instability and breast cancer susceptibility

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.     

A murine model of Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.     

Differing responses of Nijmegen breakage syndrome and ataxia telangiectasia cells to ionizing radiation

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G(0)/G(1) phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G(1)-phase arrest. Irradiated AT cells showed no potentially lethal damage and no G(1)-phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21(Waf1)) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.     

Heat-induced phosphorylation of NBS1 in human skin fibroblast cells

NBS1 is known to be involved in DNA damage-induced cellular responses after exposure to ionizing radiation (IR). Phosphorylation of NBS1 contributes to cell-cycle checkpoints. The aim of this study was to determine whether heat exposure induces or stimulates cellular responses mediated by the phosphorylation of NBS1 in human skin fibroblast cell lines. The results of immunofluorescent staining and Western blot analysis showed that NBS1 proteins are phosphorylated after exposure to heat in the nucleus of a normal skin fibroblast cell line (82-6 cells). This suggests that the NBS1-mediated signal transduction could be induced by heat. We further examined whether a deficiency in the NBS1 protein modifies heat sensitivity in human skin fibroblast cell lines. A skin fibroblast cell line (Gmtert), derived from a Nijmegen breakage syndrome (NBS) patient containing mutant NBS1, showed higher sensitivity to heat than the same cell line transfected with the wild-type copy of the NBS1 gene. We also showed that transfection of a DNA cassette expressing small interference RNA (siRNA) targeted to NBS1 into 82-6 cells enhanced cell sensitivity to heat. These results suggest that NBS1 is involved in cellular responses to DNA damage which is induced by heat exposure as well as by radiation exposure in human skin fibroblast cells.     

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.     

Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells

Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB). On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers. To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins. Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM. Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells. Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage.     

Correlation between the clonogenic initial slope and the response of polykaryon-forming units: the behavior of strains defective in XRCC5 and ATM and the heritability of small variations in radioresponse

The polykaryon-forming unit (PFU) assay measures the survival of multiple cycles of DNA synthesis after exposure to ionizing radiation, and it is known that there is a strong correlation between the slope of the PFU dose-response curve and the clonogenic initial slope. This suggests that DNA lesions expressed in clonogens are also important in PFU. Cells having a mutation in XRCC5 (also known as Ku80; strain xrs-6) and ATM (strain AT5BIVA) were hypersensitive in the PFU assay and in clonogens, while a strain of xrs-6 cells transfected with hamster wild-type XRCC5 cDNA displayed wild-type resistance in both assays. These data suggest that the DNA double-strand break (DSB) is an important lesion in PFU, although the relative radioresistance of PFU compared to clonogens indicates differential DSB toxicity. We propose that this results from the absence of cytokinesis-related loss of DNA fragments. Small variations in the radioresponse of PFU were observed between CHO K1 cell substrains, such that the xrs parental substrain RR-CHOK1 (carrying wild-type XRCC5) was more sensitive than an independent K1 substrain (E-CHOK1). Somatic hybridization showed that this variation is heritable and that the resistant E phenotype is dominant. In RR-CHOK1 cells there was a biphasic PFU radioresponse, which suggests that there may be transient expression at a locus selectively affecting PFU sensitivity.     

Cellular and genetic studies in three UV-sensitive Chinese hamster mutants

Three UV sensitive (UV^s) mutants (CHO43RO, CHO423PV, CHO30PV), characterized by different levels of reduction in their ability to perform unscheduled DNA synthesis (UDS), were analysed for spontaneous and UV-induced frequency of chromosomal aberrations and for sensitivity to alkylating agents. The baseline frequency of chromosomal aberrations was in the normal range, whereas after UV irradiation a positive correlation between the degree of UV sensitivity and the rate of chromosomal breakage was observed. Survival experiments after mutagen exposure indicated that the UV^s clones are characterized by different levels of hypersensitivity to bifunctional alkylating agents whereas the sensitivity to monofunctional alkylating agents is in the normal range. Genetic analysis performed by measuring the survival after UV in hybrids produced by fusing UV^s cells with wild-type or UV^s cells belonging to the six Chinese hamster complementation groups, indicated that the three clones carry recessive mutations and belong to c.g. 2. These findings suggest that defects in the same gene may result in different degrees of phenotypic alterations.Abbreviations CG complementation group - EMS ethyl methane sulfonate - MMS methyl methane sulfonate - MMC mitomycin C - UV ultraviolet - UDS unscheduled DNA synthesis     

ATM blocks tunicamycin-induced endoplasmic reticulum stress

Endoplasmic reticulum stress (ER-stress) is associated with ataxia telangiectasia mutated (ATM) gene. We present here conclusive data showing that ATM blocks ER-stress induced by tunicamycin or ionizing radiation (IR). X-box protein-1 (XBP-1) splicing, GRP78 expression and caspase-12 activation were increased by tunicamycin or IR in Atm-deficient AT5BIVA fibroblasts. Activation of caspase-12 and caspase-3 by tunicamycin was significantly reduced in cells transfected with wild-type Atm (AT5BIVA/wtATM). Atm knockdown by siRNA, however, noticeably elevated ER-stress and chemosensitivity to tunicamycin. In summary, we present substantial data demonstrating that ATM blocks the ER stress signaling associated with cancer cell proliferation.     

Targeted disruption of NBS1 reveals its roles in mouse development and DNA repair

Nijmegen breakage syndrome (NBS) is an autosomal recessive hereditary disease that shares some common defects with ataxia-telangiectasia. The gene product mutated in NBS, named NBS1, is a component of the Mre11 complex that is involved in DNA strand-break repair. To elucidate the physiological roles of NBS1, we disrupted the N-terminal exons of the NBS1 gene in mice. NBS1(m/m) mice are viable, growth retarded and hypersensitive to ionizing radiation (IR). NBS1(m/m) mice exhibit multiple lymphoid developmental defects, and rapidly develop thymic lymphoma. In addition, female NBS1(m/m) mice are sterile due to oogenesis failure. NBS1(m/m) cells are impaired in cellular responses to IR and defective in cellular proliferation. Most systematic and cellular defects identified in NBS1(m/m) mice recapitulate those in NBS patients, and are essentially identical to those observed in Atm(-/-) mice. In contrast to Atm(-/-) mice, spermatogenesis is normal in NBS1(m/m) mice, indicating that distinct roles of ATM have differential requirement for NBS1 activity. Thus, NBS1 and ATM have overlapping and distinct functions in animal development and DNA repair.