Induction of HSPA4 and HSPA14 by NBS1 overexpression contributes to NBS1-induced in vitro metastatic and transformation activity

Background  

Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the MRN complex, a central player associated with double-strand break (DSB) repair. We previously demonstrated that NBS1 overexpression contributes to transformation through the activation of PI 3-kinase/Akt. NBS1 overexpression also induces epithelial-mesenchymal transition through the Snail/MMP2 pathway.     

Nbs1 is required for ATR-dependent phosphorylation events

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.     

Nijmegen breakage syndrome: The clearance pathway for mutant nibrin protein is allele specific

The autosomal recessive disorder Nijmegen breakage syndrome (NBS) is caused by mutations in the NBN gene which codes for the protein nibrin (NBS1; p95). In the majority of cases, a 5 bp deletion, a founder mutation, leads to a hypomorphic 70 kD protein, p70-nibrin, after alternative initiation of translation. Protein levels are of relevance for the clinical course of the disease, particularly with regard to malignancy. Here, mechanisms and efficiency of mutant protein clearance were examined in order to establish whether these have an impact on nibrin abundance. Cell lines from NBS patients and retroviral transductants were treated with proteasome and lysosome inhibitors and examined by semi-quantitative immunoblotting for p70-nibrin and p95-nibrin levels. The results show that p70-nibrin is degraded by the proteasome with varying efficiency in cell lines from different NBS patients leading to lower or higher steady state levels of this partially active protein fragment. In contrast, a previously described NBN missense mutation, which disturbs protein folding due to the substitution of a critical arginine by tryptophan, was found to be cleared by lysosomal microautophagy leading also to lower cellular levels. The data show that truncated nibrin and misfolded nibrin have different clearance pathways.     

Ataxia-telangiectasia, an evolving phenotype

Ataxia-telangiectasia (A-T) is a progressive neurodegenerative disorder, with onset in early childhood and a frequency of approximately 1 in 40,000 births in the United States. A-T is seen among all races and is most prominent among ethnic groups with a high frequency of consanguinity. The syndrome includes: progressive cerebellar ataxia, dysarthric speech, oculomotor apraxia, choreoathetosis and, later, oculocutaneous telangiectasia. Immunodeficiency with sinopulmonary infections, cancer susceptibility (usually lymphoid), and sensitivity to ionizing radiation are also characteristic. Laboratory findings include: (1) elevated alphafetoprotein (AFP), (2) cerebellar atrophy on magnetic resonance imaging, (3) reciprocal translocations between chromosomes 7 and 14 in lymphocytes, (4) absence or dysfunction of the ATM protein, (5) radiosensitivity, as demonstrated by colony survival assay (CSA), and (6) mutations in the ATM gene. The latter are usually truncating or splicing mutations; approximately 10% are missense mutations. Mutations are found across the entire gene. Almost all recurring mutations are found on unique haplotypes that represent founder effects and ancestral relationships between patients. In addition to radiosensitivity and sensitivity to radiomimetic chemicals, the phenotype of A-T cells includes defective damage-induced activation of the cell cycle checkpoints at G1, S and G2/M. With the aid of molecular testing, A-T can now be distinguished from other autosomal recessive cerebellar ataxias (ARCAs) such as Friedreich ataxia, Mre11 deficiency (AT-like disease), and the oculomotor apraxias 1 (aprataxin deficiency) and 2 (senataxin deficiency). Other "A-T variants" include: (1) Nijmegen breakage syndrome (NBS) or nibrin/Nbs1 deficiency, with microcephaly and mental retardation but without ataxia, apraxia, or telangiectasia, and 2) A-T(Fresno), a phenotype that combines features of both NBS and A-T, with mutations in the ATM gene. The term "A-T variant" has a diminishing usefulness.     

Chk2 Activation Dependence on Nbs1 after DNA Damage

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.     

Extreme variation in apoptosis capacity amongst lymphoid cells of Nijmegen breakage syndrome patients

The human genetic disorder, Nijmegen breakage syndrome (NBS), is characterised by radiosensitivity, immunodeficiency and an increased risk for cancer, particularly lymphoma. The NBS1 gene codes for a protein, nibrin, involved in the processing/repair of DNA double strand breaks and in cell cycle checkpoints. The majority of patients (>90%) are homozygous for a founder mutation. Despite this genetic homogeneity, the syndrome shows considerable clinical variability, for example, in age at development of a malignancy. We hypothesised that one reason for such variation might be individual differences in the clearance of heavily damaged precancerous cells by apoptosis. To test this hypothesis we have examined a set of 30 lymphoblastoid B-cell lines from NBS patients for their capacity to enter into apoptosis after a DNA-damaging treatment. There was a substantial 40-fold variation in apoptosis between cell lines from different patients. NBS patient cell lines could be grouped into a large, apoptosis-deficient group and a smaller group with essentially normal apoptotic response to DNA damage. In both groups, cell lines were proficient in TP53 phosphorylation and stabilisation after the same DNA-damaging treatment. Thus the observed variation in apoptosis capacity is not due to failure to activate TP53. Despite the large variation in apoptosis, no statistically significant correlation between apoptotic capacity of patient cell lines and clinical course of the disease was apparent.     

Detection of a tandem BRCT in Nbs1 and Xrs2 with functional implications in the DNA damage response

MOTIVATION: Human Nbs1 and its homolog Xrs2 in Saccharomyces cerevisiae are part of the conserved MRN complex (MRX in yeast) which plays a crucial role in maintaining genomic stability. NBS1 corresponds to the gene mutated in the Nijmegen breakage syndrome (NBS) known as a radiation hyper-sensitive disease. Despite the conservation and the importance of the MRN complex, the high sequence divergence between Nbs1 and Xrs2 precluded the identification of common domains downstream of the N-terminal Fork-Head Associated (FHA) domain. RESULTS: Using HMM-HMM profile comparisons and structure modelling, we assessed the existence of a tandem BRCT in both Nbs1 and Xrs2 after the FHA. The structure-based conservation analysis of the tandem BRCT in Nbs1 supports its function as a phosphoserine binding domain. Remarkably, the 5 bp deletion observed in 95% of NBS patients cleaves the tandem at the linker region while preserving the structural integrity of each BRCT domain in the resulting truncated gene products.     

Rescue of a telomere length defect of Nijmegen breakage syndrome cells requires NBS and telomerase catalytic subunit.

Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.     

The Mre11 complex and ATM: collaborating to navigate S phase

Recently, findings regarding a group of cancer predisposition and chromosome instability syndromes, Nijmegen breakage syndrome (NBS), the ataxia-telangiectasia-like disorder (A-TLD) and ataxia telangiectasia have shed light on the unexpected role of recombinational DNA repair proteins in DNA-damage-dependent cell-cycle regulation. Mutations in the Mre11 complex cause A-TLD and NBS. In addition, functions of the Mre11 complex have been biochemically linked to ATM, the large protein kinase that is defective in ataxia-telangiectasia cells by the observation that Nbs1 is a bona fide substrate of the ATM kinase.     

Independent roles for nibrin and Mre11-Rad50 in the activation and function of Atm

The Atm protein kinase and Mre11-Rad50-nibrin (MRN) complex play an integral role in the cellular response to DNA double-strand breaks. Mutations in Mre11 and nibrin result in the radiosensitivity disorders ataxia-telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS), respectively. Cells from ATLD and NBS patients are deficient in activation of the Atm protein kinase and phosphorylation of downstream Atm targets following irradiation. However, the roles of individual MRN complex proteins in Atm function are not clear, because the mutations in NBS and ATLD cells result in global effects on the MRN complex. Previously we showed that the C-terminal 100 amino acids of nibrin were necessary and sufficient to translocate the MRN complex to the nucleus. Here we have taken advantage of this feature of nibrin to create isogenic cell lines lacking either nibrin or Mre11-Rad50 in the nucleus. We found that nuclear expression of Mre11-Rad50, but not nibrin, stimulated Atm activation at early times after low doses of radiation. At later times or higher doses of irradiation, Atm activation was independent of Mre11-Rad50 or nibrin. The requirement of MRN complex proteins for downstream Atm phosphorylation events following irradiation was more complex. Phosphorylation of nibrin and Chk2 by Atm required Mre11-Rad50 expression in the nucleus at early times after irradiation, reflecting the stimulation of Atm activation by Mre11-Rad50. By contrast, autophosphorylation of Chk2 and phosphorylation of Smc1 at Ser-957 was dependent on the MRN complex 60 min after irradiation, even though Atm was activated at that time point. These results indicate an independent role for Mre11-Rad50 in the activation of Atm and suggest nibrin and/or Mre11-Rad50 also act as adaptors for some downstream Atm phosphorylation events.     

The gene for the ataxia-telangiectasia variant,Nijmegen breakage syndrome,maps to a 1-cM interval on chromosome 8q21.

Nijmegen breakage syndrome (NBS; Seemanová II syndrome) and Berlin breakage syndrome (BBS), also known as ataxia-telangiectasia variants, are two clinically indistinguishable autosomal recessive familial cancer syndromes that share with ataxia-telangiectasia similar cellular, immunological, and chromosomal but not clinical findings. Classification in NBS and BBS was based on complementation of their hypersensitivity to ionizing radiation in cell-fusion experiments. Recent investigations have questioned the former classification into two different disease entities, suggesting that NBS/BBS is caused by mutations in a single radiosensitivity gene. We now have performed a whole-genome screen in 14 NBS/BBS families and have localized the gene for NBS/BBS to a 1-cM interval on chromosome 8q21, between markers D8S271 and D8S270, with a peak LOD score of 6.86 at D8S1811. This marker also shows strong allelic association to both Slavic NBS and German BBS patients, suggesting the existence of one major mutation of Slavic origin. Since the same allele is seen in both former complementation groups, genetic homogeneity of NBS/BBS can be considered as proved.     

Nibrin forkhead-associated domain and breast cancer C-terminal domain are both required for nuclear focus formation and phosphorylation

The Mre11.Rad50.nibrin protein complex plays an essential role in the mammalian cellular response to DNA double-strand breaks. The disorder Nijmegen breakage syndrome (NBS) results from mutations in the NBS1 gene that encodes nibrin, and NBS cells are radiosensitive and defective in S-phase checkpoint activation following irradiation. In response to radiation, nibrin is phosphorylated by Atm, and the Mre11.Rad50.nibrin complex relocalizes to form punctate nuclear foci. The N terminus of nibrin contains a forkhead-associated (FHA) domain and a breast cancer C-terminal (BRCT) domain, the functions of which are unclear. To determine the role of the FHA and BRCT domains in nibrin function, we have performed site-directed mutagenesis of conserved residues in these motifs. Mutations in the nibrin FHA and BRCT domains did not affect interaction with Mre11.Rad50 or nuclear localization of the complex. However, mutation of conserved residues in either domain disrupted nuclear focus formation and blocked nibrin phosphorylation after irradiation, suggesting that these events may be functionally interdependent. Despite an effect on nibrin phosphorylation, expression of the FHA or BRCT mutants in NBS cells restored the downstream phosphorylation of Chk2 and Smc1, necessary for S-phase checkpoint activation. None of the mutations revealed separate functions for the FHA or BRCT domains, suggesting they do not function independently.     

Werner syndrome protein associates with gamma H2AX in a manner that depends upon Nbs1

The WRN protein is mutated in the chromosomally unstable Werner syndrome (WS) and the Nbs1 protein is mutated in Nijmegen breakage syndrome (NBS). The Nbs1 protein is an integral component of the M/R/N complex. Although WRN is known to interact with this complex in response to gamma-irradiation, the mechanism of action is unclear. Here, we show that WRN co-localizes and associates with gamma H2AX, a marker protein of DNA double strand breaks (DSBs), after cellular exposure to gamma-irradiation. While the DNA damage-inducible Nbs1 foci formation is normal in WS cells, WRN focus formation is defective in NBS cells. Consistent with this, gamma H2AX colocalizes with Nbs1 in WS cells but not with WRN in NBS cells. The defective WRN-gamma H2AX association in NBS cells can be complemented with wild-type Nbs1, but not with an Nbs1 S343A point mutant that lacks an ATM phosphorylation site. WRN associates with H2AX in a manner dependent upon the M/R/N complex. Our results suggest a novel pathway in which Nbs1 may recruit WRN to the site of DNA DSBs in an ATM-dependent manner.     

Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization

The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.     

Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt.     

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.     

Screening of Nijmegen breakage syndrome 1 mutations in four unrelated families by polymerase chain reaction using sequence-specific primers

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by a marked predisposition to lymphoreticular malignancies. The rarity of the disease and the presence, in several cases, of a mild clinical phenotype make diagnosis difficult. The underlying gene, NBS1, consists of 16 exons and encodes nibrin, a member of the hMRE11/hRAD50/hNBS1 protein complex. In addition to the "Slavic mutation," 657del5, identified in more than 100 patients with NBS, 9 other mutations have been found in families of different ethnic origin. We have developed a polymerase chain reaction (PCR) method to rapidly detect the private mutations, 742insGG and 835del4, in exon 7 and the 900del25 mutation in exon 8 of the NBS1 gene. In particular, we designed NBS1-specific primers for wild-type and mutated alleles, and optimized a specific PCR protocol for each mutation. We used this method to analyze 4 unrelated NBS families, 3 from Italy and 1 from Morocco. We believe it could be a useful tool for: (1) confirming the NBS diagnosis in the presence of clinical signs of the disease; (2) identifying NBS heterozygotes and performing prenatal diagnosis in families with affected members; and (3) screening selected populations in which the frequency of NBS might be higher because of a founder effect.     

Genetic Mapping Using Microcell-Mediated Chromosome Transfer Suggests a Locus for Nijmegen Breakage Syndrome at Chromosome 8q21-24

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by microcephaly, short stature, immunodeficiency, and a high incidence of cancer. Cultured cells from NBS show chromosome instability, an increased sensitivity to radiation-induced cell killing, and an abnormal cell-cycle regulation after irradiation. Hitherto, patients with NBS have been divided into the two complementation groups V1 and V2, on the basis of restoration of radioresistant DNA synthesis, suggesting that each group arises from a different gene. However, the presence of genetic heterogeneity in NBS has been considered to be controversial. To localize the NBS gene, we have performed functional complementation assays using somatic cell fusion between NBS-V1 and NBS-V2 cells, on the basis of hyper-radiosensitivity, and then have performed a genomewide search for the NBS locus, using microcell-mediated chromosome transfer followed by complementation assays based on radiosensitivity. We found that radiation resistance was not restored in the fused NBS-V1 and NBS-V2 cells and that only human chromosome 8 complements the sensitivity to ionizing radiation, in NBS cell lines. In complementation assays performed after the transfer of a reduced chromosome, merely the long arm of chromosome 8 was sufficient for restoring the defect. Our results strongly suggest that NBS is a homogeneous disorder and that the gene for NBS is located at 8q21-24.     

Dual functions of Nbs1 in the repair of DNA breaks and proliferation ensure proper V(D)J recombination and T-cell development

Immunodeficiency and lymphoid malignancy are hallmarks of the human disease Nijmegen breakage syndrome (NBS; OMIM 251260), which is caused by NBS1 mutations. Although NBS1 has been shown to bind to the T-cell receptor alpha (TCRα) locus, its role in TCRβ rearrangement is unclear. Hypomorphic mutations of Nbs1 in mice and patients result in relatively mild T-cell deficiencies, raising the question of whether the truncated Nbs1 protein might have clouded a certain function of NBS1 in T-cell development. Here we show that the deletion of the entire Nbs1 protein in T-cell precursors (Nbs1(T-del)) results in severe lymphopenia and a hindrance to the double-negative 3 (DN3)-to-DN4 transition in early T-cell development, due to abnormal TCRβ coding and signal joints as well as the functions of Nbs1 in T-cell expansion. Chromatin immunoprecipitation (ChIP) analysis of the TCR loci reveals that Nbs1 depletion compromises the loading of Mre11/Rad50 to V(D)J-generated DNA double-strand breaks (DSBs) and thereby affects resection of DNA termini and chromatin conformation of the postcleavage complex. Although a p53 deficiency relieves the DN3→DN4 transition block, neither a p53 deficiency nor ectopic expression of TCRαβ rescues the major T-cell loss in Nbs1(T-del) mice. All together, these results demonstrate that Nbs1's functions in both repair of V(D)J-generated DSBs and proliferation are essential for T-cell development.