Atm-deficient mice: a paradigm of ataxia telangiectasia

A murine model of ataxia telangiectasia was created by disrupting the Atm locus via gene targeting. Mice homozygous for the disrupted Atm allele displayed growth retardation, neurologic dysfunction, male and female infertility secondary to the absence of mature gametes, defects in T lymphocyte maturation, and extreme sensitivity to gamma-irradiation. The majority of animals developed malignant thymic lymphomas between 2 and 4 months of age. Several chromosomal anomalies were detected in one of these tumors. Fibroblasts from these mice grew slowly and exhibited abnormal radiation-induced G1 checkpoint function. Atm-disrupted mice recapitulate the ataxia telangiectasia phenotype in humans, providing a mammalian model in which to study the pathophysiology of this pleiotropic disorder.     

Radiation-activated DNA-binding protein constitutively present in ataxia telangiectasia nuclei

We have recently described the appearance of a specific DNA-binding protein in nuclei from human cells exposed to ionizing radiation which was not detected in nuclear extracts from unperturbed cells (Singh, S. P., and Lavin, M. F. (1990) Mol. Cell. Biol. 10, 5279-5285). We report here a similar activity which is constitutively present in nuclei of both unirradiated and irradiated cells from patients with the human genetic disorder ataxia telangiectasia (A-T). Activity was present in unirradiated nuclear extracts from 3 A-T cell lines of different complementation groups, but was not detected or was present only at a low level in 4 controls. Active protein was detected in the cytoplasm of both cell types. Exposure to ionizing radiation did not change the amount of DNA binding activity in A-T nuclei but led to an increase in nuclei from 4 control cell lines. Purification of the binding activities from A-T nuclei and control cytoplasm was carried out by affinity chromatography, as described previously for control extracts (Teale, B., Singh, S. P., Khanna, K. K., Findik, D., and Lavin, M. F. (1992) J. Biol. Chem. 267, 10295-10301). Southwestern analysis and UV cross-linking confirmed the presence of a major DNA-binding species at 70 kDa in both cases with a minor binding activity at 47 kDa also evident. It was not possible to distinguish between the binding activities from A-T and control cells under different conditions, and phosphorylation was required for binding activity in both cases. Footprint analysis revealed that the same sequence was being recognized by the control and A-T proteins. The constitutive presence of a specific radiation-responsive DNA-binding protein in A-T cells may be indicative of a continuous state of stress in these cells.     

High frequency and error-prone DNA recombination in ataxia telangiectasia cell lines

The only specific DNA repair defect found in ataxia telangiectasia (A-T) cells is mis-repair of cleaved DNA. In this report we measured DNA recombination, given its role in DNA repair and genetic instability. Using plasmids containing selectable reporter genes, we found a higher frequency of both chromosomal recombination (>100 times) and extra-chromosomal recombination (27 times) in SV40-transformed A-T cell lines compared with in an SV40-transformed normal fibroblast cell line. Southern analysis of single A-T colonies exhibiting post-integration recombination revealed that 24/27 had undergone aberrant rearrangements; recombination in normal fibroblast colonies was achieved by gene conversion in 8/11 clones and 10/11 clones showed unchanged copies of the plasmid. Using co-transfection of two integrating plasmids, each containing a separate deletion in the xgprt reporter gene, the 27 times difference in extra-chromosomal recombination was found when the plasmids were cleaved at a distance from the reporter gene. When the plasmids were cleaved within the reporter gene, the co-transfection frequency was reduced in A-T, but was increased in normal cells. We conclude that A-T cell lines have not only a high frequency chromosomal and extra-chromosomal recombination, but also exhibit error-prone recombination of cleaved DNA.     

Evolution of chromosomal abnormalities in sequential cytogenetic studies of ataxia telangiectasia

Sequential cytogenetic studies of four patients with ataxia telangiectasia showed the progressive development of lymphocyte clones, each marked with a rearranged chromosome 14. Initial studies had shown random chromosomal breaks and rearrangements. Later studies in all patients showed nonrandom rearrangement of chromosome 14 with a breakpoint at 14q12 and with the distal segment translocated to either chromosome 14 or 7. The proportion of circulating lymphocytes carrying the marker tended to increase with time, accounting for the majority of the lymphocytes eventually in one case. The marked lymphocyte clones evolved further, as a result of loss of the small centric portions of the rearranged chromosome 14 (14pter leads to 14q12). Perhaps the abnormal clones in ataxia telangiectasia escape immunologic surveillance and flourish in an immunologically impaired environment. Subsequent to the loss of the centric portion of the rearranged chromosome 14, the cells may acquire additional capabilities that enhance malignant transformation.     

Defective control of apoptosis, radiosensitivity, and spindle checkpoint in ataxia telangiectasia

We examined the regulation of apoptosis, radiosensitivity, and spindle checkpoint in response to DNA-damaging agents in ataxia telangiectasia (AT)-derived lymphoblastoid cell lines (AT-LCLs), which lack AT mutated (ATM) protein expression. In addition to the previous findings that AT-LCLs are defective in regulation of cell cycle at the G1, S, and G2-M checkpoints in response to X-ray irradiation (X-IR) and are highly sensitive to X-IR (J. Biol. Chem., 271: 20486-20493, 1996), we showed for the first time that AT-LCLs were defective in X-IR-associated spindle checkpoint control. The cells were also resistant to early apoptosis as much as LCLs derived from patients with Li-Fraumeni syndrome (LFS-LCLs). Terminal deoxynucleotidyl transferase-mediated nick end labeling assay of LCLs, however, demonstrated a significant increase in apoptotic cells among AT-LCLs cultured over a longer period after X-IR. These findings were in contrast to those of LFS-LCL, which showed very little increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive population, even in cells with hyperploidy. Thus, although early apoptosis and cell cycle controls in response to DNA damage are disrupted in both ATM and p53 mutations, cells from AT patients are much more susceptible to late-onset apoptosis than those of LFS. These differences may depend on the level of accumulation of DNA damage and/or threshold that triggers late-onset cell death in ATM or p53 mutations. Our findings allow a better understanding of the role of ATM in p53-dependent and independent signal transduction pathways in response to DNA damaging agents.     

Possible control of cell death pathways in ataxia telangiectasia. A case report

Peripheral blood monocytes (PBMCs) obtained from a boy with the neuroimmunodegenerative syndrome of ataxia telangiectasia (AT) failed to aggregate or replicate efficiently when mitogenically activated under serum-depleted conditions. These cells rapidly swelled, then slowly shrank, and flattened as they excreted vesicles containing chromatin. This accelerated cell death with loss of homoadhesiveness could be prevented in vitro in most of the homozygous PBMCs by adding large amounts of autologous serum or by adding mixtures of Th1 cytokines, serum factors, and redox agents. However, even in high-serum media containing added cytokines, 20-30% of the homozygous PBMCs quickly flattened, produced minicells, and died. Since the defective functions of the human ataxia-telangiectasia nuclear kinase gene (ATM) could be bypassed in vitro in these defective AT PMBCs by addition of appropriate cytokines and redox survival factors, it may be possible to slow the progressive losses of ATM-deficient lymphoid cells seen in vivo. Since the neuronal degeneration in AT, as seen in the retrovirus-induced neuroimmunodegenerative syndromes, may also be a consequence of impairment of the central and peripheral immune system, it may become possible to prevent the neurodegeneration in AT by using signaling therapies that upregulate the ATM-induced signal deficiencies in the developing immune system.     

Chylothorax: an update

Chylothorax is occasionally found in malignant disease and following sympathectomy, but is seen more frequently after cardiothoracic surgery. The varied anatomy of the thoracic duct, limited individual experience and a lack of appreciation of the factors indicating surgical intervention have led to controversy regarding its optimal management. This article reviews historical aspects, clinical features and guidelines for conservative and surgical management.     

Coccidioidomycosis: an update

The incidence of coccidiodomycosis in the southwestern United States has sharply increased over the last decade as a result of environmental and demographic changes. It is important to review the epidemiology, diagnostic indicators, and therapeutic options for both immunocompetent and immunocompromised patients who have a particularly high risk of infection.     

Poly(ADP-ribose) polymerase activity is not affected in ataxia telangiectasia cells and knockout mice

Poly(ADP-ribose) polymerase (PARP) is a constitutive factor of the DNA damage surveillance network in dividing cells. Based on its capacity to bind to DNA strand breaks, PARP plays a regulatory role in their resolution in vivo. ATM belongs to a large family of proteins involved in cell cycle progression and checkpoints in response to DNA damage. Both proteins may act as sensors of DNA damage to induce multiple signalling pathways leading to activation of cell cycle checkpoints and DNA repair. To determine a possible relationship between PARP and ATM, we examined the PARP response in an ATM-null background. We demonstrated that ATM deficiency does not affect PARP activity in human cell lines or Atm-deficient mouse tissues, nor does it alter PARP activity induced by oxidative damage or gamma-irradiation. Our results support a model in which PARP and ATM could be involved in distinct pathways, both effectors transducing the damage signal to cell cycle regulators.     

Chromosomes and causation of human cancer and leukemia. XLVIII. T-cell acute leukemia in ataxia telangiectasia

Cytogenetic and immunologic studies were performed on the cells of an 18-year-old female with ataxia telangiectasia (AT) associated with acute lymphocytic leukemia (ALL). At the onset of the leukemia 15.4% of peripheral blood cells stimulated with phytohemagglutinin (PHA) contained a tandem translocation of the long arm of chromosome #14, i.e., t(14;14). To ascertain if these karyotypically abnormal cells and the leukemic cells had a common lineage, chromosome analyses were performed on bone marrow cells. Examination of the marrow cells on the seven occasions when leukemic cells were present in the marrow, including times when they were predominant, showed only a normal karyotype without the presence of t(14;14). However, an abnormal clone, which had the karyotype 45,XX,-9,t(9;6)(q12;q13), was identified in the marrow cells on the last examination during the terminal phase of the leukemia. Immunologically, the ALL was classified as an atypical type which had characteristics in common with certain T-cell subsets. We suggest that the malignant cells did not originate from the preexisting cells with a tandem duplication of the 14q.     

KARP-1 is induced by DNA damage in a p53- and ataxia telangiectasia mutated-dependent fashion

The KARP-1 (Ku86 Autoantigen Related Protein-1) gene, which is expressed from the human Ku86 autoantigen locus, appears to play a role in mammalian DNA double-strand break repair as a regulator of the DNA-dependent protein kinase complex. Here we demonstrate that KARP-1 gene expression is significantly up-regulated following exposure of cells to DNA damage. KARP-1 mRNA induction was completely dependent on the ataxia telangiectasia and p53 gene products, consistent with the presence of a p53 binding site within the second intron of the KARP-1 locus. These observations link ataxia telangiectasia, p53, and KARP-1 in a common pathway.     

Analgesia in cancer: beliefs and an update

Substance P (SP) appears to mediate many processes of the central nervous system, including pain. This report deals with modulation of opioid binding in the mouse brain by SP and SP fragments, as well as by salts and guanine nucleotides. Binding studies of the selective mu opioid receptor agonist [D-Ala2, MePhe4,Gly(ol)5]enkephalin (DAMGO) to mouse brain membrane preparations demonstrated that guanine nucleotide modulation of DAMGO binding affinity was modified by SP. However, SP had little or no influence on inhibition of DAMGO binding induced by salts, such as MgCl2, CaCl2, or NaCl. By replacing GTP with GppNHp, SP (0.1 nM) produced multiple affinity forms of the DAMGO receptor, while at a higher concentration (10 nM), SP lost its influence on DAMGO binding. Furthermore, 0.1 nM SP changed DAMGO binding parameters in a medium containing NaCl, CaCl2, and GppNHp such that the high- and low-affinity conformations of the receptor converted to a single site following the addition of SP to the incubation medium. While the C-terminal SP fragment SP(5-11) was without effect, the N-terminal SP fragments SP(1-9) and SP(1-7) appeared to imitate SP in modifying GppNHp-modulated DAMGO binding. These results suggest that SP functions as a modulator of opioid binding at the mu receptor and it appears that the N-terminus of SP plays a role in the modulatory process.     

Social phobia: an update

The literature on social phobia is reviewed in this article. Social phobia has undergone considerable diagnostic evolution to reach its present form in DSM-IV. Its differential diagnosis includes panic disorder with agoraphobia, avoidant personality disorder, depression, and "shyness." Cross-cultural issues are important to consider because the disorder may manifest differently in different cultures and social settings. It is common, with a lifetime prevalence of 13.3% in the United States according to recent epidemiological studies. Underrecognition of social phobia remains an issue of concern. Comorbidity with other psychiatric disorders, including other anxiety disorders, depression, alcohol abuse, and personality disorders, frequently occurs. Current conceptualizations of the etiology of social phobia reflect psychodynamic theories and evidence from family and genetic studies, neurobiological research, and neuroimaging. Drugs such as monoamine oxidase inhibitors, selective serotonin-reuptake inhibitors, benzodiazepines, and beta3-adrenergic blockers have proven to be efficacious, as has cognitive-behavioral treatment, including group approaches.