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.     

NBS1, the Nijmegen breakage syndrome gene product, regulates neuronal proliferation and differentiation

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder, characterized by progressive microcephaly, growth retardation, immunodeficiency, and pre-disposition to tumor formation. To investigate the functions of the NBS gene product, NBS1, on neurons, PC12 cells overexpressing NBS1 and related mutants and primary cortical neuronal culture were used in the present study. Small interfering RNA (siRNA) was applied to repress the expression of endogenous Nbs1 in PC12 cells and primary cortical neurons. We demonstrated that overexpression of NBS1 increases cellular proliferation and decreases the apoptosis of PC12 cells in serum withdrawal and ionizing irradiation, through the activation of phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Overexpression of NBS1 also decreases neurite elongation on PC12 cells under nerve growth factor stimulation. Transfection of NBS1-overexpressing PC12 cells with a dominant negative Akt mutant attenuates the neuroprotection and cellular proliferation effects of NBS1 while having no effect on neurite elongation. PC12 cells overexpressing NBS657del5 and NBS653 mutants, in which the major NBS1 protein in cells are truncated proteins, have decreased cellular proliferation, increased cell death, and decreased neurite elongation compared with those of control PC12 cells. Repression of Nbs1 by siRNA decreases the PI 3-kinase activity and Akt phosphorylation levels, and induces neurite elongation in PC12 cells even without nerve growth factor stimulation. Repression of Nbs1 by siRNA in primary cortical neurons also increased neurite elongation, but increased neuronal death. We conclude that NBS1 can regulate neuronal proliferation and neuroprotection via PI 3-kinase/Akt pathway while regulating neuronal differentiation in a different pathway. Excessive accumulation of truncated protein secondary to 657del5 mutation may be detrimental to neurons, leading to defective neuronal proliferation and differentiation.     

Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive human disease whose clinical features include growth retardation, immunodeficiency, and increased susceptibility to lymphoid malignancies. Cells from NBS patients exhibit gamma-irradiation sensitivity, S-phase checkpoint defects, and genomic instability. Recently, it was demonstrated that this chromosomal breakage syndrome is caused by mutations in the NBS1 gene that result in a total loss of full-length NBS1 expression. Here we report that in contrast to the viability of NBS patients, targeted inactivation of NBS1 in mice leads to early embryonic lethality in utero and is associated with poorly developed embryonic and extraembryonic tissues. Mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture, and this finding suggests that NBS1 mediates essential functions during proliferation in the absence of externally induced damage. Together, our results indicate that the complex phenotypes observed in NBS patients and cell lines may not result from a complete inactivation of NBS1 but may instead result from hypomorphic truncation mutations compatible with cell viability.     

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.     

657del5 mutation of the Nijmegen breakage syndrome gene (NBS1) in the Turkish population

The 657del5 mutation of the NBS1 gene has been demonstrated in most patients with Nijmegen breakage syndrome (NBS). We identified four Turkish families in which probands were diagnosed as having NBS and found to be homozygous for the 657del5 mutation. The 657del5 allele in the four Turkish families had a single origin.     

ATFresno: a phenotype linking ataxia-telangiectasia with the Nijmegen breakage syndrome.

This report describes twin girls with typical features of ataxia-telangiectasia, including increased alpha-fetoprotein, radio-resistant DNA synthesis, characteristic chromosome abnormality, and immunodeficiency. They have, in addition, microcephaly and mental retardation. Complementation studies were performed utilizing Sendai virus--mediated fusion of fibroblast cell lines. Complementation was observed with patients in ataxia-telangiectasia complementation groups A, C, and E but not with the cell line from a patient with the Nijmegen breakage syndrome, in which patients have microcephaly, radio-resistant DNA synthesis, chromosome aberrations, and immunodeficiency but lack ataxia and telangiectasia. These data suggest that the Nijmegen breakage syndrome and the patients described here are not genetically distinct entities but form a spectrum of one disorder.     

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.     

The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.     

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.     

DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells

NBS1-deficient cells exhibit pronounced radiosensitivity and defects in chromosome integrity after ionizing radiation (IR) exposure, yet show only a minor defect in DNA double-strand break (DSB) rejoining, leaving an as yet unresolved enigma as to the nature of the radiosensitivity of these cells. To further investigate the relationship between radiosensitivity, DSB repair, and chromosome stability, we have compared cytological and molecular assays of DSB misrejoining and repair in NBS1-defective, wild type, and NBS1-complemented cells after IR damage. Our findings suggest a subtle defect in overall DSB rejoining in NBS1-defective cells and uniquely also reveal reduced ability of NBS1-defective cells to rejoin correct ends of DSBs. In agreement with published results, one of two different NBS1-defective cell lines showed a slight defect in overall rejoining of DSBs compared to its complemented counterpart, whereas another NBS line did not show any difference from wild type cells. Significant defects in the correct rejoining of DSBs compared to their respective controls were observed for both NBS1-defective lines. The defect in DSB rejoining and the increased misrejoining detected at the molecular level were also reflected in higher levels of fragments and translocations, respectively, at the chromosomal level. This work provides both molecular and cytological evidence that NBS1-deficient cells have defects in DSB processing and reveals that these molecular events can be manifest cytologically.     

The catalytic subunit of DNA-dependent protein kinase regulates proliferation, telomere length, and genomic stability in human somatic cells

The DNA-dependent protein kinase (DNA-PK) complex is a serine/threonine protein kinase comprised of a 469-kDa catalytic subunit (DNA-PK_cs) and the DNA binding regulatory heterodimeric (Ku70/Ku86) complex Ku. DNA-PK functions in the nonhomologous end-joining pathway for the repair of DNA double-stranded breaks (DSBs) introduced by either exogenous DNA damage or endogenous processes, such as lymphoid V(D)J recombination. Not surprisingly, mutations in Ku70, Ku86, or DNA-PK_cs result in animals that are sensitive to agents that cause DSBs and that are also immune deficient. While these phenotypes have been validated in several model systems, an extension of them to humans has been missing due to the lack of patients with mutations in any one of the three DNA-PK subunits. The worldwide lack of patients suggests that during mammalian evolution this complex has become uniquely essential in primates. This hypothesis was substantiated by the demonstration that functional inactivation of either Ku70 or Ku86 in human somatic cell lines is lethal. Here we report on the functional inactivation of DNA-PK_cs in human somatic cells. Surprisingly, DNA-PK_cs does not appear to be essential, although the cell line lacking this gene has profound proliferation and genomic stability deficits not observed for other mammalian systems.     

Effects of cisplatin on telomerase activity and telomere length in BEL—7404 human hepatoma cells

INTRODUCTIONPrimary hepatocellular carcinoma (PHCC), oneof the most common malignancies in the world, isan aggressive cancer. The mean survival time fromestablishment of diagnosi8 is only about 4 months(2 months if the diagnosis is made 1ate). It causesab…     

Purification of human telomerase complexes identifies factors involved in telomerase biogenesis and telomere length regulation

The identities and roles of proteins associated with human telomerase remain poorly defined. To gain insight, we undertook an affinity purification of endogenously assembled human telomerase complexes. We show that specific subsets of H/ACA, Sm, and hnRNP proteins associate with active and inactive telomerase RNPs, while two NTPase proteins associate preferentially with active enzyme. All three core H/ACA-motif binding proteins are telomerase holoenzyme components essential for RNP accumulation. On the other hand, telomerase RNPs lacking interaction with Sm proteins or hnRNP C remain fully functional for telomere elongation. Curiously, overexpression of either associated hnRNP protein (hnRNP C and hnRNP U) or either NTPase protein (NAT10 and GNL3L) induced telomere shortening. Our findings suggest that endogenous human telomerase complexes are more heterogeneous than those of single-celled eukaryotes, have predominantly shared rather than telomerase-specific proteins, and make numerous regulatory interactions.     

Replication proteins influence the maintenance of telomere length and telomerase protein stability

We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells. Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.     

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.     

Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.     

The analysis of telomere length and telomerase activity in cloned pigs and cows

Inefficiency in the production of cloned animals is most likely due to epigenetic reprogramming errors after somatic cell nuclear transfer (SCNT). In order to investigate whether nuclear reprogramming restores cellular age of donor cells after SCNT, we measured telomere length and telomerase activity in cloned pigs and cattle. In normal pigs and cattle, the mean telomere length was decreased with biological aging. In cloned or transgenic cloned piglets, the mean telomere length was elongated compared to nuclear donor fetal fibroblasts and age-matched normal piglets. In cloned cattle, no increases in mean telomere length were observed compared to nuclear donor adult fibroblasts. In terms of telomerase activity, significant activity was observed in nuclear donor cells and normal tissues from adult or new-born pigs and cattle, with relatively higher activity in the porcine tissues compared to the bovine tissues. Cloned calves and piglets showed the same level of telomerase activity as their respective donor cells. In addition, no difference in telomerase activity was observed between normal and transgenic cloned piglets. However, increased telomerase activity was observed in porcine SCNT blastocysts compared to nuclear donor cells and in vitro fertilization (IVF)-derived blastocysts, suggesting that the elongation of telomere lengths observed in cloned piglets could be due to the presence of higher telomerase activity in SCNT blastocysts. In conclusion, gathering from the comparative studies with cattle, we were able to demonstrate that telomere length in cloned piglets was rebuilt or elongated with the use of cultured donor fetal fibroblasts.