Apurinic DNA endonuclease activities in repair-deficient human cell lines.

Several autosomal recessive diseases are associated with apparent DNA repair defects in cell culture. It seemed likely that a defect in excision repair reported for ataxia telangiectasia cells might reflect a lack of apurinic endonuclease activity. We report here normal levels of apurinic endonuclease activity in extracts of cell lines derived from patients with ataxia telangiectasia, xeroderma pigmentosum (complementation group D), Cockayne dwarfism, Fanconi anemia and Bloom syndrome.     

Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.     

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts.

Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal.     

An enzyme activity in normal and ataxia telangiectasia cell lines which is involved in the repair of gamma-irradiation-induced DNA damage.

An enzyme that enhances the activity of DNA polymerase I (EC 2.7.7.7) for gamma-irradiated calf thymus DNA was demonstrated in cellular extracts of normal human fibroblasts and lymphoid-cell lines. This enzyme was found to be deficient in all cellular extracts of fibroblasts and lymphoid-cell lines examined from patients with the autosomal recessive disease ataxia telangiectasia. The activity in cellular extracts from normal fibroblasts was removed when heated to 100 degrees C for 2 min or when the assay was performed at 4 degrees C. No significant deficiency in primer-activating enzyme activity was observed in cell-free extracts of lymphoid lines from patients with xeroderma pigmentosum, Huntington's chorea or neurofibromatosis, or from an ataxia telangiectasia heterozygote.     

Effect of aphidicolin on de novo DNA synthesis,DNA repair and cytotoxicity in γ-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase α and consequently of de novo DNA synthesis in human cells. We report here that in γ-irradiated normal human cells, aphidicolin (at 5 μg/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. γ-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase α is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.     

Gene dosage and complementation analysis of ataxia telangiectasia lymphoblastoid cell lines assayed by induced chromosome aberrations

An approach of general applicability to mammalian radiosensitive mutants has been used in the analysis of gene dosage and complementation in ataxia telangiectasia (A-T). Thymidine residues in DNA of one parental lymphoblastoid cell line were substituted with bromodeoxyuridine before fusion with a second parental cell line, to allow differential staining of the two sets of chromosomes. Following gamma-irradiation, induced chromosome aberrations were scored in diploid and homokaryon cells from each parental line as well as in heterokaryons. Four complementation groups were ascertained among 7 A-T cell lines. Analysis of heterokaryons formed between appropriate combinations of normal, A-T homozygote and A-T heterozygote cells, gave a quantitative measure of gene dosage and demonstrated increasing radiosensitivity with increasing numbers of A-T alleles.     

Effect of aphidicolin on de novo DNA synthesis, DNA repair and cytotoxicity in gamma-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase alpha and consequently of de novo DNA synthesis in human cells. We report here that in gamma-irradiated normal human cells, aphidicolin (at 5 micrograms/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. gamma-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase alpha is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.     

G2-phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndrome

Abstract. The relationship between G₂-phase checkpoint activation, cytoplasmic cyclin-B1 accumulation and nuclear phosphorylation of p34^CDC2 was studied in Nijmegen breakage syndrome cells treated with DNA damaging agents. Experiments were performed on lymphoblastoid cell lines from four Nijmegen breakage syndrome patients with different mutations, as well as on cells from an ataxia telangiectasia patient. Lymphoblastoid cell lines were irradiated with 0.50–2 Gy X-rays and the percentage of G₂-phase accumulated cells was evaluated by means of flow cytometry in samples that were harvested 24 h later. The G₂-checkpoint activation was analysed by scoring the mitotic index at 2 and 4 h after treatment with 0.5 and 1 Gy X-rays and treatment with the DNA double-strand break inducer calicheamicin-γ1. Cytoplasmic accumulation of cyclin-B1 was evaluated by means of fluorescence immunostaining or Western blotting, in cells harvested shortly after irradiation with 1 and 2 Gy. The extent of tyrosine 15-phosphorylated p34^CDC2 was assessed in the nuclear fractions. Nijmegen breakage syndrome cells showed suboptimal G₂-phase checkpoint activation respect to normal cells and were greatly different from ataxia telangiectasia cells. Increased cytoplasmic cyclin-B1 accumulation was detected by both immunofluorescence and immunoblot in normal as well as in Nijmegen breakage syndrome cells. Furthermore, nuclear p34^CDC2. phosphorylation was detected at a higher level in Nijmegen breakage syndrome than in ataxia telangiectasia cells. In conclusion, our data do not suggest that failure to activate checkpoints plays a major role in the radiosensitivity of Nijmegen breakage syndrome cells.     

Influence of ataxia telangiectasia gene dosage on bleomycin-induced chromosome breakage and inhibition of replication in human lymphoblastoid cell lines

Long-term lymphoblastoid cell lines, obtained by E-B virus transformation of peripheral blood lymphocytes, retain many of the features of hypersensitivity to environmental agents found in primary cultures and fibroblast strains from patients with genetic diseases. Primary lymphocyte cultures from patients with ataxia telangiectasia, a cancer-prone genetic disease, have increased sensitivity to chromosomal damage induced by the radio-mimetic drug, bleomycin. In order to study the expression of ataxia telangiectasia gene dosage in lymphoblastoid cell lines, we examined chromosomal aberrations in lines containing two, one, or no alleles for ataxia telangiectasia. These were derived from ataxia telangiectasia homozygotes, from ataxia telangiectasia obligate heterozygotes, and from presumably normal donors, respectively. Chromosome preparations were made 46 h after a 2 h exposure to bleomycin and scored for chromosome breakage, for the relative rate of cell replication as assessed by sister chromatid differentiation patterns, and for the frequency of sister chromatid exchanges. Baseline frequencies of chromosome breakage and sister chromatid exchanges, and baseline rates of cell replication were similar in all nine lymphoblastoid cell lines. Following treatment with 25 or 250 mU/ml bleomycin, all the lymphoblastoid cell lines showed increased chromosome breakage and decreased cell replication. The lymphoblastoid cell lines from the ataxia telangiectasia homozygotes had significantly increased chromosome breakage and decreased rate of cell replication after either bleomycin dose in comparison with the normal or with the ataxia telangiectasia heterozygous lines. Sister chromatid exchange frequencies were not altered by bleomycin exposure.     

The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells.

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.     

Residual ataxia telangiectasia mutated protein function in cells from ataxia telangiectasia patients, with 5762ins137 and 7271T-->G mutations, showing a less severe phenotype

We have assessed several ataxia Telangiectasia mutated (ATM)-dependent functions in cells derived from ataxia telangiectasia patients, carrying either an ATM 5762ins137 splice site or a 7271T-->G missense mutation, with a less severe phenotype compared with the classical disorder. ATM kinase in vitro, from 5762ins137 cells, showed the same specific activity as ATM in normal cells, but the protein was present at low levels. In contrast, mutant ATM kinase activity in the 7271T-->G cells was only about 6% that of the activity in normal cells, although the level of mutant protein expressed was similar to normal cells. Phosphorylation of the DNA double strand break repair proteins Nbs1 and hMre11, following DNA damage, was observed in normal and 7271T-->G cells but was almost absent in both 5762ins137 and classical ataxia telangiectasia cells. The kinetics of p53 response was intermediate between normal and classical ataxia telangiectasia cells in both the 7271T-->G and 5762ins137 cells, but interestingly, c-Jun kinase activation following DNA damage was equally deficient in cell lines derived from all the ataxia telangiectasia patients. Our results indicate that levels of ATM kinase activity, but not induction of p53 or c-Jun kinase activity, in these cells correlate with the degree of neurological disorder in the patients.     

Influence of ATM function on interactions between telomeres and nuclear matrix

The ATM (ataxia telangiectasia mutated) gene product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control. The human ATM protein shows similarity to several yeast and mammalian proteins involved in meiotic recombination and cell cycle progression. Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia (AT), influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in AT cells is due to altered interactions between the telomeres and the nuclear matrix. These interactions were examined in nuclear matrix halos prior to and after irradiation. A difference was observed in the ratio of soluble and matrix-associated telomeric DNA between cells derived from AT and normal individuals. Treatment with ionizing radiation affected the ratio of soluble and matrix-associated telomeric DNA only in the AT cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, such interactions were examined in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix seen in AT cells. Fibroblasts from AT individuals transfected with a wild-type ATM gene had corrected telomere-nuclear matrix interactions. In experiments designed to determine whether there is a link between the altered telomere-nuclear matrix interactions and defective telomere movement and clustering, a significant difference was observed in the ratio of soluble compared to matrix-associated telomeric DNA sequences in meiocytes of Atm(-/-) and control mice. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix and that alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene. This paper summarizes our recent publications on the influence of inactivation of ATM on the interaction of telomeres with nuclear matrix in somatic and germ cells.     

The effect of anions and cations on cardiac adenylate cyclase: interactions with isoprenaline and GTP

The level of actin was found to decrease markedly when ataxia telangiectasia lymphoblastoid cells were stepped from low to high density culture conditions. Additionally, as the actin levels decreased the levels of a protein species of 37K daltons increased by orders of magnitude. Partial proteolytic digestion of the 37K protein and actin revealed that the primary structures of these proteins were not related. This phenomena was observed in three out of four ataxia telangiectasia lymphoblastoid cell lines but not in lymphoblastoid cells derived from normal individuals.     

An evaluation of the Drosophila zeste somatic eye mutation test for the detection of genotoxic azo dyes and an aromatic amine

The rate of DNA synthesis was studied in normal cell strain and in strains from patients suffering from inherited disorder ataxia telangiectasia (AT). After exposure to reactively low doses of oxic X-rays (0–4 krad) DNA synthesis was depressed in AT cell strains to a significantly lesser extent than in normal cells. This response was observed in both an “excision-deficient” and an “excision-proficient” strain. In contrast, there was no difference in DNA-synthesis inhibition between AT and normal cells after UV exposure. After X-irradiation of cells from patients with xeroderma pigmentosum, both complementation group A and XP variants, the observed rate of DNA synthesis was equal to that in normal cells. An exception was the strain XP3BR which has been shown to be X-ray sensitive. This strain exhibited diminished DNA synthesis inhibition after X-ray doses below 1 krad.These data suggest a relationship between hypersensitivity to X-rays and diminished depression of DNA synthesis.     

ATMIN defines an NBS1-independent pathway of ATM signalling

The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM co-localised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling.     

毛细血管扩张性共济失调突变蛋白在细胞凋亡过程中被Caspase┐3降解

新近的研究揭示：ｃａｓｐａｓｅ蛋白酶在细胞凋亡中起着死亡执行者的重要功能．一些蛋白相继被证明在细胞凋亡中可被ｃａｓｐａｓｅ特异切割,其中参与ＤＮＡ损伤修复过程的聚ＡＤＰ核糖聚合酶（ＰＡＲＰ）以及ＤＮＡ依赖的蛋白激酶（ＤＮＡ－ＰＫ）,在细胞凋亡过程中被ｃａｓｐａｓｅ选择性切割具有特殊的功能意义．为探索与ＤＮＡ－ＰＫ催化亚基有较高同源性,含有ｃａｓｐａｓｅ切割位点,且功能上目前也被认为是感受ＤＮＡ损伤和参与信号传导途径的ＡＴＭ（Ａｔａｘｉａｔｅｌａｎｇ－ｉｅｃｔａｓｉａｍｕｔａｔｅｄ）蛋白,是否在凋亡过程中也可被切割而降解？应用体外转录与翻译系统获得ＡＴＭ蛋白的ＰＩ３Ｋ结构域,同时通过建立无细胞反应体系获得含ｃａｓｐａｓｅ活性的细胞抽提液,将两者在体外共同保温．结果发现：ＡＴＭ蛋白与ｃａｓｐａｓｅ－３能免疫共沉淀,ＡＴＭ蛋白的ＰＩ３Ｋ结构域可被ｃａｓｐａｓｅ－３特异切割,并观察到辐射诱发细胞调亡中ＡＴＭ蛋白的降解．从而进一步证实了ＤＮＡ损伤修复的抑制,促进细胞凋亡的发生．     

Radiation, DNA damage and cancer

The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasia and Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk of developing cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders provide valuable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role in sporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (ATM) has recently been shown to be a tumour suppressor gene in T-cell prolymphocytic leukaemia, and there is increasing evidence that individuals with one mutated ATM or Nijmegen breakage syndrome (NBS1) allele have an increased predisposition to cancer.     

Oxidative stress in ataxia telangiectasia

Ataxia telangiectasia is one of a group of recessive hereditary genomic instability disorders and is characterized by progressive neurodegeneration, immunodeficiency and cancer susceptibility. Heterozygotes for the mutated gene are more susceptible to cancer and to ischaemic heart disease. The affected gene, ATM (ataxia telangiectasia mutated), has been cloned and codes for a protein kinase (ATM), which orchestrates the cellular response to DNA double-strand breaks after ionising radiation. An underlying feature of ataxia telangiectasia is oxidative stress and there is chronic activation of stress response pathways in tissues showing pathology such as the cerebellum, but not in the cerebrum or liver. ATM has also been shown to be activated by insulin and to have a wider role in signal transduction and cell growth. Many, but not all, aspects of the phenotype can be attributed to a defective DNA damage response. The oxidative stress may result directly from accumulated DNA damage in affected tissues or ATM may have an additional role in sensing/modulating redox homeostasis. The basis for the observed tissue specificity of the oxidative damage in ataxia telangiectasia is not clear.