Altered telomere nuclear matrix interactions and nucleosomal periodicity in ataxia telangiectasia cells before and after ionizing radiation treatment.
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Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions 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 similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene. (+info)
Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM.
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The human neurodegenerative and cancer predisposition condition ataxia-telangiectasia is characterized at the cellular level by radiosensitivity, chromosomal instability, and impaired induction of ionizing radiation-induced cell cycle checkpoint controls. Recent work has revealed that the gene defective in ataxia-telangiectasia, termed ATM, encodes an approximately 350-kDa polypeptide, ATM, that is a member of the phosphatidylinositol 3-kinase family. We show that ATM binds DNA and exploit this to purify ATM to near homogeneity. Atomic force microscopy reveals that ATM exists in two populations, with sizes consistent with monomeric and tetrameric states. Atomic force microscopy analyses also show that ATM binds preferentially to DNA ends. This property is similar to that displayed by the DNA-dependent protein kinase catalytic subunit, a phosphatidylinositol 3-kinase family member that functions in DNA damage detection in conjunction with the DNA end-binding protein Ku. Furthermore, purified ATM contains a kinase activity that phosphorylates serine-15 of p53 in a DNA-stimulated manner. These results provide a biochemical assay system for ATM, support genetic data indicating distinct roles for DNA-dependent protein kinase and ATM, and suggest how ATM may signal the presence of DNA damage to p53 and other downstream effectors. (+info)
Solid tissues removed from ATM homozygous deficient mice do not exhibit a mutator phenotype for second-step autosomal mutations.
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The presence of increased frequencies of blood-derived and solid tumors in ataxia-telangiectasia (A-T) patients, coupled with a role for the ATM (A-T mutation) protein in detecting specific forms of DNA damage, has led to the assumption of a mutator phenotype in A TM-deficient cells. Supporting this assumption are observations of increased rates of chromosomal aberrations and intrachromosomal homologous recombinational events in the cells of A-T patients. We have bred mice with knockout mutations for the selectable Aprt (adenine phosphoribosyltransferase) locus and the Atm locus to examine the frequency of second-step autosomal mutations in Atm-deficient cells. Two solid tissues were examined: (a) the ear, which yields predominately mesenchymal cells; and (b) the kidney, which yields predominately epithelial cells. We report here the lack of a mutator phenotype for inactivating autosomal mutations in solid tissues of the Atm-deficient mice. (+info)
Ataxia telangiectasia mutated deficiency affects astrocyte growth but not radiosensitivity.
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The cancer-prone neurodegenerative disorder, ataxia telangiectasia (A-T), results from mutations of ATM (ataxia telangiectasia mutated). Individuals with A-T are also hypersensitive to ionizing radiation (IR). Cultured cells from A-T individuals or Atm-/- mice have cell cycle and growth defects and are generally considered radiosensitive. However, it has been shown recently that cell populations in the Atm-/- central nervous system are radioresistant. To define specific IR sensitivities of neural populations, we analyzed Atm-/- astrocytes. Here we show that Atm-/- astrocytes exhibit premature senescence, express constitutively high levels of p21, and have impaired p53 stabilization. However, in contrast to radiosensitive Atm-/- fibroblasts and radioresistant Atm-/- neurons, survival of Atm-/- astrocytes after IR was similar to wild-type astrocytes. Additionally, p53-null astrocytes, but not fibroblasts, were moderately more radioresistant than their wild-type counterparts, suggesting that the deficit in p53 stabilization observed in Atm-null cells is not a measure of radiation susceptibility. Thus, in astrocytes, the function of Atm in cellular growth and radiosensitivity is distinct. These data may have implications for ATM disruption strategies as a radiosensitizing treatment for brain tumors. (+info)
Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks.
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The Brca1 (breast cancer gene 1) tumor suppressor protein is phosphorylated in response to DNA damage. Results from this study indicate that the checkpoint protein kinase ATM (mutated in ataxia telangiectasia) was required for phosphorylation of Brca1 in response to ionizing radiation. ATM resides in a complex with Brca1 and phosphorylated Brca1 in vivo and in vitro in a region that contains clusters of serine-glutamine residues. Phosphorylation of this domain appears to be functionally important because a mutated Brca1 protein lacking two phosphorylation sites failed to rescue the radiation hypersensitivity of a Brca1-deficient cell line. Thus, phosphorylation of Brca1 by the checkpoint kinase ATM may be critical for proper responses to DNA double-strand breaks and may provide a molecular explanation for the role of ATM in breast cancer. (+info)
Lack of involvement of ataxia telangiectasia mutated (ATM) in regulation of nuclear factor-kappaB (NF-kappaB) in human diploid fibroblasts.
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It has been suggested that the cellular response to exposure to ionizing radiation involves activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and that this response is defective in cells from individuals with ataxia telangiectasia (AT). In one study, it was found that SV40 large T-transformed cells derived from a patient null for the AT mutated (ATM) gene exhibited constitutive activation of NF-kappaB and that in those cells, inhibition of NF-kappaB by expression of a modified form of IkappaBalpha led to correction of the radiosensitivity associated with the AT phenotype [M. Jung et al., Science (Washington DC), 268: 1691-1621, 1995]. From those data, it was suggested that NF-kappaB played a role in the AT phenotype. We show here that normal diploid cells derived from AT patients do not exhibit constitutive activation of NF-kappaB. Furthermore, we provide data that the transformation process associated with SV40 large T antigen expression in AT-/- cells leads to aberrant cellular responses. Our studies highlight the importance of using diploid, nontransformed AT-/- cells for in vitro studies relevant to the AT phenotype whenever possible. (+info)
Somatic rearrangement of chromosome 14 in human lymphocytes.
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Ataxia-telangiectasia is a rare genetic disorder associated with immune deficiency, chromosome instability, and a predisposition to lymphoid malignancy. We have detected chromosomally anomalous clones of lymphocytes in eight patients with this disorder. Chromosome banding disclosed that the clones are consistently marked by structural rearrangement of the long arm (q) of chromosome 14. A translocation involving 14q was found in clones obtained from seven of the eight patients whereas a ring 14 chromosome was found in a clone obtained from the other. These findings as well as data obtained by others for patients with ataxia-telangiectasia suggest that structural rearrangement of 14q is the initial chromosomal change in lymphocyte clones of patients with this disorder. Chromosomes of lymphocytes from one of the patients were studied before and after the onset of chronic lymphocytic leukemia. Before leukemia was diagnosed, the patient had a lymphocyte clone with a 14q translocation. This clone appears to have given rise to the leukemic cells. We hypothesize that structural rearrangement of 14q is directly related to abnormal growth of lymphocytes and that it may be a step toward the development of lymphoid malignancies. Increasing evidence, provided by others, for the nonrandom involvement of 14q in African-type Burkitt's lymphoma and other lymphoid neoplasms further strengthens this hypothesis. (+info)
Heterozygous germline ATM mutations do not contribute to radiation-associated malignancies after Hodgkin's disease.
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PURPOSE: The successful treatment of Hodgkin's disease has been associated with an increased incidence of secondary malignancies. To investigate whether genetic factors contribute to the development of secondary tumors, we collected family cancer histories and performed mutational analysis of the ataxia-telangiectasia (AT) gene, ATM, in a cohort of Hodgkin's disease survivors with secondary malignancies. ATM was chosen for evaluation because of the increased radiosensitivity of cells derived from AT patients and obligate heterozygotes and the epidemiologic observation that AT carriers are at increased risk for radiation-induced breast cancer. PATIENTS AND METHODS: Fifty-two patients who developed one or more neoplasms after treatment for Hodgkin's disease participated in this study. Personal and family histories of cancer were obtained through patient interviews and review of medical records. ATM mutational analysis was performed using a yeast-based protein truncation assay. RESULTS: Seventy-six secondary neoplasms were observed in this cohort of 52 Hodgkin's disease survivors, with 18 patients (35%) developing more than one secondary neoplasm. Positive family histories of cancer were present in 11 (21%) of 52 patients, compared with three (4%) of 68 Hodgkin's disease patients in a comparison cohort who did not develop secondary neoplasms (P =.008; Fisher's exact test). No germline ATM mutations were identified, resulting in an estimated AT carrier frequency in this population of 0% (90% confidence interval, 0% to 4%). CONCLUSION: Analysis of the number of tumors per individual and the family history of cancer in our cohort suggests that genetic factors may contribute to development of secondary neoplasms in a subset of Hodgkin's disease survivors. Mutational analysis, however, does not support a significant role for heterozygous truncating ATM mutations. Future studies evaluating other genes involved in DNA damage response pathways are warranted. (+info)