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PubMed2001

Ataxia telangiectasia mutated-dependent apoptosis after genotoxic stress in the developing nervous system is determined by cellular differentiation status.

Ataxia-telangiectasia (A-T) is a neurodegenerative syndrome resulting from dysfunction of ATM (ataxia telangiectasia mutated). The molecular details of ATM function in the nervous system are unclear, although the neurological lesions in A-T are probably developmental because they appear during childhood. The nervous systems of Atm-null mice show a pronounced defect in apoptosis that is induced by DNA damage, suggesting that ATM may function to eliminate DNA-damaged neurons. Here we show that Atm-dependent apoptosis occurs at discrete stages of neurogenesis. Analysis of gamma-irradiated mouse embryos showed that Atm-dependent apoptosis occurred only in the postmitotic populations that were present in the neuroepithelial subventricular zone of the developing nervous system. Notably, Atm deficiency did not prevent radiation-induced apoptosis in multipotent precursor cells residing in the proliferating ventricular zone. Atm-dependent apoptosis required p53 and coincided with the specific phosphorylation of p53 and caspase-3 activation. Thus, these data show that Atm functions early in neurogenesis and underscore the selective requirement for Atm in eliminating damaged postmitotic neural cells. Furthermore, these data demonstrate that the differentiation status of neural cells is a critical determinant in the activation of certain apoptotic pathways.

Animals↗
PubMed1993

Hyperbaric oxygen inhibits the growth of cultured rabbit lens epithelial cells without affecting glutathione level.

Studies on human patients and experimental animals indicate that hyperbaric O2 can opacify the lens nucleus and damage the lens epithelium in vivo. Here we investigate the effects of hyperbaric O2 on cultured rabbit lens epithelial cells (LECs). When the cells were exposed to 50 atm O2 (99% O2 + 1% CO2) for 3 hr there were no immediate effects on morphology, viability and transport processes (uptake of 86Rb and 14C-alpha AIB). In addition, the O2 treatment did not lower the high level of reduced glutathione or increase the low level of oxidized glutathione. However, 50 atm O2 did produce a near doubling in the glycolytic rate which maintained ATP at levels only slightly lower than normal. Although the 3-hr O2 treatment was not lethal, it completely inhibited cell division for 2 days. After 2 days, growth was initiated and, at day 7 the rate of growth was faster than the controls (control cells were treated with ambient air or 50 atm N2 for 3 hr). Cells treated with 8 atm O2 for 3 hr exhibited a slowed rate of growth, relative to controls, while exposure to 2 atm O2, did not inhibit mitosis. Changes in morphology (multilayering and elongation) of cells exposed to 50 atm O2, but not the controls, were evident 7 days after the 3-hr exposure. The incorporation of [35S]methionine into individual polypeptides and [3H]thymidine into DNA was significantly inhibited immediately following a 3-hr treatment with 50 atm O2, but both parameters recovered within 2 days. DNA strand breaks were observed in LECs following hyperbaric O2 treatment as low as 4 atm O2 for 3 hr and increased with higher pressures of O2, but not N2. Treatment with 50 atm O2 nearly doubled the activity of the DNA repair enzyme, poly-ADP-ribose polymerase, and decreased the level of its substrate NAD+; the latter effect was reduced by 3-aminobenzamide, an inhibitor of the enzyme. Thus, although LECs tolerated brief exposures to high pressures of O2 without cell death, DNA damage occurred at relatively low pressures of O2. All of the effects of hyperbaric O2 on LECs occurred without any alteration of the normal levels of reduced and oxidized glutathione. It appears that GSH is important in maintaining cell viability during exposure to an elevated level of O2, but that it is incapable of preventing O2-induced effects on growth and DNA.

Animals↗
PubMed1991

Repair of helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins.

The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.

Adenosine Triphosphatases↗
PubMed2000

Increased sensitivity to chromatid aberration induction by bleomycin and neocarzinostatin results from alterations in a DNA damage response pathway.

DNA damage response pathways coordinate the cellular response to DNA damage. To investigate the roles of tumor suppressor genes in these pathways, human lymphoblastoid cells (wild-type, p53-/-, ATM-/-) were treated for 1 h with 0-3 microg/ml of the radiomimetic compound bleomycin (BLM), and cells treated in G(2) were analyzed for chromatid aberrations. BLM-induced aberration frequencies were significantly increased, to the greatest extent in the ATM-/- cells and, to a lesser extent, in the p53-/- cells compared to wild-type cells. These observations are consistent with p53 and ATM acting in a damage response pathway activated by DNA strand breaks. The consequences of disrupting this pathway were further investigated by studies using wortmannin, a PI-3 kinase and DNA repair inhibitor. Wortmannin significantly increased the BLM-induced aberration frequencies in all but the ATM-/- cells, elevating the sensitivity of p53-/- cells to ATM-/- levels and that of wild-type cells to intermediate levels. These differential sensitivities suggest that the ATM phenotype is the result of dual cellular defects, one involving p53 and the other a wortmannin-sensitive component. Similar studies in Brca1+/- and Brca2+/- human lymphoblasts showed no increased sensitization to BLM in the absence of inhibitor, and differential sensitization by wortmannin. To determine if there was any substrate specificity for p53- and ATM-mediated DNA damage responses, chromatid aberrations were assessed in wild-type, p53-/-, and ATM-/- cells exposed to 0-0.4 microg/ml neocarzinostatin (NCS) for 1 h. In contrast to results with BLM, the p53-/- cells exhibited a low sensitivity to NCS-induced aberrations, similar to wild-type, while ATM-/- cells remained highly sensitive. This suggests that the response to BLM- and NCS-induced lesions involves different mechanisms.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A.

Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.

Ataxia Telangiectasia↗
PubMed2000

Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia.

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and down-regulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Sp1 decreased. A causal relationship was established between these observations by demonstrating that up-regulation of Sp1 DNA binding activity by granulocyte/macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells can be explained by induction of ATM protein and kinase activity with time post-irradiation. Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

Activation of ataxia telangiectasia-mutated DNA damage checkpoint signal transduction elicited by herpes simplex virus infection.

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection.

Acid Anhydride Hydrolases↗
PubMed2006

Aurothiomalate inhibits transformed growth by targeting the PB1 domain of protein kinase Ciota.

We recently identified the gold compound aurothiomalate (ATM) as a potent inhibitor of the Phox and Bem1p (PB1)-PB1 domain interaction between protein kinase C (PKC) iota and the adaptor molecule Par6. ATM also blocks oncogenic PKCiota signaling and the transformed growth of human lung cancer cells. Here we demonstrate that ATM is a highly selective inhibitor of PB1-PB1 domain interactions between PKCiota and the two adaptors Par6 and p62. ATM has no appreciable inhibitory effect on other PB1-PB1 domain interactions, including p62-p62, p62-NBR1, and MEKK3-MEK5 interactions. ATM can form thio-gold adducts with cysteine residues on target proteins. Interestingly, PKCiota (and PKCzeta) contains a unique cysteine residue, Cys-69, within its PB1 domain that is not present in other PB1 domain containing proteins. Cys-69 resides within the OPR, PC, and AID motif of PKCiota at the binding interface between PKCiota and Par6 where it interacts with Arg-28 on Par6. Molecular modeling predicts formation of a cysteinyl-aurothiomalate adduct at Cys-69 that protrudes into the binding cleft normally occupied by Par6, providing a plausible structural explanation for ATM inhibition. Mutation of Cys-69 of PKCiota to isoleucine or valine, residues frequently found at this position in other PB1 domains, has little or no effect on the affinity of PKCiota for Par6 but confers resistance to ATM-mediated inhibition of Par6 binding. Expression of the PKCiota C69I mutant in human non-small cell lung cancer cells confers resistance to the inhibitory effects of ATM on transformed growth. We conclude that ATM inhibits cellular transformation by selectively targeting Cys-69 within the PB1 domain of PKCiota.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Role of DNA-dependent protein kinase in the process of radiation-induced aberration formation.

PURPOSE: To further investigate the role of DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutation (ATM) in the formation of chromosome aberrations, we analysed radiation-induced aberrations in M059J cells, complemented with the PRKDC (DNA-PK) gene by introducing a fragment of human chromosome 8 containing a copy of the human PRKDC gene. One hybrid cell line (M059J Fus1) displayed kinase activity and was radioresistant; the other hybrid cell line (M059J Fus9) showed no kinase activity and was radiosensitive. Both Fus1 and Fus9 cells have only a low ATM activity. Wortmannin, an inhibitor of DNA-PKCS and ATM, was added before irradiation in order to study the effect of DNA-PKCS--and ATM--inhibition on the formation of chromosome aberrations. Furthermore, aberration formation was studied in a lymphoblastoid ATM-deficient cell line AT-1 and in an ATM-proficient control. MATERIALS AND METHODS: Confluent cells were irradiated with 200 kV X-rays. Dicentrics, excess acentric fragments, chromatid/ isochromatid breaks and chromatid exchanges were scored in the absence and presence of wortmannin. RESULTS: In M059J-Fus1 cells and normal lymphoblastoid cells, only chromosome-type aberrations were observed independently of the presence of wortmannin. In DNA-PK-deficient Fus9 cells and in ATM-deficient AT-1 cells, an increasing proportion (30-80%) of cells containing chromatid-type aberrations was observed. This proportion increased with irradiation dose and with wortmannin addition. The aberration yields observed in the complemented M059J-Fus1 cell line were much lower than the corresponding yields observed in the deficient M059J and AT-1 cell lines. However, the low yields observed in the DNA-PK-proficient 'wild-type' cell line M059K were not completely restored. CONCLUSIONS: Since in M059J-Fus1 cells the radioresistant phenotype with respect to chromosome-type aberration formation was restored by the complementation of PRKDC, ATM expression determines the chromosomal radiosensitivity of M059J cells only to a minor extent. The increasing presence of chromatid-type aberrations in cells irradiated in G0/G1 phase as observed either in DNA-PK- or ATM-deficient cells definitely requires the lack of either kinase. Thus, the aberration spectrum observed is determined by the genetic profile of the respective cells and aberration class amplitudes can be modulated by the inhibition of either kinase.

Androstadienes↗
PubMed2001

Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure.

STUDY DESIGN: This in vitro study clarifies the role of nitric oxide (NO) in human lumbar intervertebral disc metabolism. OBJECTIVE: To investigate the effects of NO on proteoglycan synthesis in human lumbar discs and to test the hypothesis that NO is a mediator of the changes in proteoglycan synthesis in response to hydrostatic pressure. SUMMARY OF BACKGROUND DATA: The authors have clarified that hydrostatic pressure has an apparent effect on proteoglycan synthesis as well as matrix metalloproteinase production in the intervertebral disc. The cellular mechanisms underlying the response of disc cells to hydrostatic pressure remain to be clarified. Herniated lumbar discs produce NO in response to interleukin (IL)-1 beta. In articular cartilage, NO mediates the change of proteoglycan synthesis by IL-1 or shear stress. METHODS: Fifty-eight lumbar intervertebral disc specimens were obtained from patients who had undergone posterior discectomy. The specimens were chopped into 1-2-mm cubes and were incubated in a plastic syringe with 1 mL Dulbecco's modified Eagle's medium (DMEM). The syringes were placed in a water-filled pressure vessel kept at 37 C. Hydrostatic pressures of 1 (control), 3, and 30 atmospheres (atm) were applied. Proteoglycan synthesis was determined from (35)S-sulfate incorporation rates. Nitrite (the stable oxidation product of NO) concentration in DMEM was determined by a spectrophotometric method based on the Griess reaction. As a competitive inhibitor of NO synthases, N(G)-methyl-l-arginine (l-NMA, 10-1000 micromol) and as an organic donor of NO, S-nitroso-N-acetylpenicillamine (SNAP, 1-200 micromol) were used. RESULTS: Addition of l-NMA suppressed NO production and increased proteoglycan synthesis rates in the intervertebral disc specimens in a dose-dependent fashion. Addition of SNAP increased exogenous NO content in the medium significantly and suppressed proteoglycan synthesis rates in a dose-dependent fashion. Three-atmosphere hydrostatic pressure stimulated the proteoglycan synthesis rates. Rates were approximately 1.3-fold greater than at 1 atm, whereas 30-atm pressure inhibited proteoglycan synthesis rates. However, the hydrostaticpressure had inverse effect on NO production. At 3 atm, NO production decreased slightly relative to 1 atm, whereas at a pressure of 30 atm, NO production was increased and was approximately 1.32-fold greater than at 1 atm. L-NMA enhanced the 3-atm pressure-induced increase in proteoglycan synthesis and also relieved the suppression of proteoglycan synthesis at a pressure of 30 atm. CONCLUSION: The current study confirmed the previous finding that human herniated lumbar disc cultures spontaneously produce NO. Endogenously generated and exogenously supplied NO inhibited proteoglycan synthesis in the intervertebral disc. Hydrostatic pressure influenced NO production by disc cells, and NO is one of the mediators that changes proteoglycan synthesis in response to hydrostatic pressure. These results may show that autocrine and paracrine mechanisms of NO play an important role in the regulation of disc cell metabolism under mechanical stress and in the pathophysiology of intervertebral disc degeneration.

Adolescent↗
PubMed1986

The effects of anaesthetics and high pressure on the responses of the rat superior cervical ganglion in vitro.

The effects of helium pressure and of general anaesthetics were studied on the responses of the isolated superior cervical ganglion of the rat, to determine how far these reflected the pressure reversal of anaesthesia seen in vivo. The method of Brown & Marsh (1974) for extracellular recording of surface potentials was adapted for use in a high-pressure chamber. Helium alone, at 130 atm, did not alter the responses of the ganglion to gamma-aminobutyric acid (GABA) but significantly depressed the depolarizing and hyperpolarizing components of the nicotinic responses, and the muscarinic responses. The potentiation of the responses to GABA caused by pentobarbitone was not altered by the application of helium, at 130 atm. This pressure also decreased further the nicotinic responses which were depressed by pentobarbitone. Nitrogen, at 34 atm (the anaesthetic ED50 in vivo) and at 68 atm, significantly decreased the nicotinic responses of the ganglia, and the addition of helium to a total of 130 atm further increased this depression. At pressures of 3.3-68 atm, nitrogen caused small decreases in the responses to GABA. Nitrous oxide at 1.5 atm (the ED50 for loss of righting reflex in mice) and at 3 atm, significantly depressed the responses to GABA and to the nicotinic agonist, but did not alter the responses to methylfurmethide. The effects of nitrous oxide were unaltered when helium was added to a total of 130 atm, although this pressure of helium added alone significantly depressed the cholinergic responses. A mixture of 50% nitrous oxide and 50% oxygen, when added to the pressure chamber, at normal atmospheric pressure, caused transient increases in the responses to GABA. The effects of temperature on GABA responses and on nicotinic responses were very different from those of pressure. Preliminary evidence suggested that raising the temperature may decrease the extent of potentiation of GABA responses by pentobarbitone. The results are discussed in relation to the pressure reversal of anaesthesia in vivo. It was concluded that there was no evidence that the basis of this interaction lay in the potentiation of GABA responses by general anaesthetics, or the depression of cholinergic responses, although the changes seen were not in all cases simply additive. It was considered that effects of general anaesthetics such as the potentiation of GABA may contribute to the effects used to measure general anaesthesia in vivo, such as loss of righting reflex, but may not be related to the non-specific actions which cause anaesthesia.

Action Potentials↗
PubMed2006

Analysis of the ataxia telangiectasia mutated-mediated DNA damage response in murine cerebellar neurons.

The DNA damage response is a network of signaling pathways that affects many aspects of cellular metabolism after the induction of DNA damage. The primary transducer of the cellular response to the double-strand break, a highly cytotoxic DNA lesion, is the nuclear protein kinase ataxia telangiectasia (A-T) mutated (ATM), which phosphorylates numerous effectors that play key roles in the damage response pathways. Loss or inactivation of ATM leads to A-T, an autosomal recessive disorder characterized by neuronal degeneration, particularly the loss of cerebellar granule and Purkinje cells, immunodeficiency, genomic instability, radiosensitivity, and cancer predisposition. The reason for the cerebellar degeneration in A-T is not clear. It has been ascribed by several investigators to cytoplasmic functions of ATM that may not be relevant to the DNA damage response. We set out to examine the subcellular localization of ATM and characterize the ATM-mediated damage response in mouse cerebellar neurons. We found that ATM is essentially nuclear in these cells and that various readouts of the ATM-mediated damage response are similar to those seen in commonly used cell lines. These include the autophosphorylation of ATM, which marks its activation, and phosphorylation of several of its downstream substrates. Importantly, all of these responses are detected in the nuclei of granule and Purkinje cells, suggesting that nuclear ATM functions in these cells similar to other cell types. These results support the notion that the cerebellar degeneration in A-T patients results from defective DNA damage response.

Animals↗
PubMed1992

Comparison of two recompression profiles in treating experimental cerebral air embolism.

The standard treatment for cerebral arterial gas embolism (CAGE) is an initial recompression to 6 atm abs on air for 30 min followed by oxygen breathing at 2.8 and 1.9 atm abs. It has been suggested that initial recompression to 2.8 atm abs on O2 may be as beneficial, thus avoiding potential treatment complications associated with the deeper depth. To test this hypothesis, we measured the recovery of the somatosensory evoked potential (SEP) following air embolism in anesthetized, ventilated cats. Air was infused into the carotid artery in increments of 0.08 ml until the SEP amplitude was reduced to less than 10% of the baseline value for 15 min. Three groups were studied. A control group (n = 10) received no further treatment after SEP suppression. The second group (6 atm abs/HBO] (n = 8) was compressed to 6 atm abs on air for 30 min followed by O2 breathing at 2.8 atm abs for 100 min. The third group (HBO) (n = 8) was compressed to 2.8 atm abs on O2 for 130 min. The control group recovered 28.8 +/- 18.2% (mean +/- SD) of the baseline amplitude, whereas the 6 atm abs/HBO group recovered 48.6 +/- 22.6%, and the HBO group recovered 62.0 +/- 20.3%. An analysis of variance revealed that only the HBO group had significantly (P less than 0.01) better recovery than the control group. There was no significant difference in SEP recovery between the 2 treatment groups. These results suggest that treating CAGE at 2.8 atm abs with O2 is a viable alternative to the current therapy.

Animals↗
PubMed1988

Exposure of rabbit lens to hyperbaric oxygen in vitro: regional effects on GSH level.

Previous studies have indicated that in vivo exposure to hyperbaric O2 may be associated with the development of nuclear cataract. In the present work, in vitro effects of hyperbaric O2 on rabbit lenses were investigated following culture of the lenses in an atmosphere of 99% O2 at pressures ranging between 1 and 100 atm. Treatment with O2 resulted in a significant decrease in the level of reduced glutathione (GSH) in the lenses even at the lower pressures studied (less than 8 atm). At 100 atm O2 the loss of GSH was 85% after a 3 hr exposure. At 8 atm O2 a significant drop in GSH concentration was shown to occur in the lens nucleus prior to loss of the tripeptide in the superficial cortex. O2-treated lenses became hazy in appearance, especially at the higher pressures, but did not become densely opaque. Pressures of N2 up to 100 atm had no effect on either lens transparency or on the concentration of GSH. Although oxidized glutathione (GSSG) was detected in the whole lens at pressures of O2 as low as 4 atm, no change in GSH level or evidence for GSSG accumulation was observed in the capsule-epithelium of the lens at pressures as high as 50 atm O2. Ninety percent of the GSSG present in lenses after exposure to 100 atm O2 could be reconverted to GSH by subsequent culture of the lenses under normal conditions. Exposure of lenses to 50 atm O2 produced a three-fold stimulation of hexose monophosphate shunt activity, equal to that which has been reported for treatment of lenses with 0.06 mM H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Effect of 12 atmospheres helium-oxygen on the response of mice to convulsant drugs.

The mechanism by which 12 atm abs of a helium-oxygen gas mixture (heliox) antagonizes behavioral effects of ethanol is unknown. Although the threshold for pressure-reversal of general anesthesia and expression of the high pressure neurologic syndrome (HPNS) is well above 12 atm abs in mice, the ethanol antagonism by 12 atm abs heliox could result from similar underlying excitatory effects. To investigate this possibility, the behavior of water-injected control mice and the latency to convulsions in drug-injected mice were determined in 1 atm abs air and 12 atm abs heliox. Four convulsant drugs were tested: picrotoxin (2 mg/kg), dl-allylglycine (300 mg/kg), isoniazid (300 mg/kg), and l-methionine-dl-sulfoximine (170 mg/kg). Responses were videotaped to observe behavior and to measure latency to the onset of myoclonus and clonus. Results indicated no observable excitatory effects of 12 atm abs in control mice. The latency to myoclonus was significantly reduced by pressure in allylglycine-treated mice but not in mice treated with the other convulsants. Latency to clonus was not significantly altered by pressure, relative to latency at 1 atm abs heliox, for any drug tested. In conclusion, the present findings indicate that exposure to 12 atm abs heliox is not proconvulsant and, thus, the findings do not support the hypothesis that 12 atm abs heliox antagonizes ethanol indirectly via an increase in central nervous system excitability.

Allylglycine↗
PubMed1998

Seventh International Workshop on Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is a rare hereditary syndrome involving cerebellar degeneration, immunodeficiency, cancer risk, and radiosensitivity. Since the cloning of the A-T gene, ATM, in 1995, research on this pleiotropic disease and its molecular basis has expanded tremendously. ATM is a large protein kinase that appears to be one of the primary sensors of DNA strand-break damage. The vast majority of mutations in ATM result in truncation and destabilization of the protein, but certain missense and splicing errors have been shown to result in a less severe phenotype. A-T heterozygotes have been shown to have a slightly increased risk of cancer, but their increased in vitro radiosensitivity does not seem to result in any in vivo sensitivity. ATM does seem to act as a classic tumor suppressor gene in T-prolymphocytic leukemia, and LOH at the ATM locus is a common event in some tumor types, suggesting a general role for ATM in cancer. Recent work has shown the interaction of ATM with proteins involved in cell cycle control, and the direct phosphorylation of some of these interactors by ATM. ATM knockout mice have been created by several groups, and recapitulate the immunodeficiency, radiosensitivity, cancer risk, and fertility defects of A-T, although the effect on the cerebellum is slight. These diverse topics, and their integration into a global understanding of A-T, were the basis of the 7th International A-T Workshop.

Animals↗
PubMed2007

Ataxia-telangiectasia mutated expression is associated with tobacco smoke exposure in esophageal cancer tissues and benzo[a]pyrene diol epoxide in cell lines.

Esophageal cancer is a substantial health problem because of its usually late stage at diagnosis and poor prognosis. Tobacco smoking and alcohol use are the most important risk factors in the development of esophageal squamous cell carcinoma (SCC). Our previous study demonstrated the binding of benzo[a]pyrene diol epoxide (BPDE), a carcinogen present in tobacco smoke and environmental pollution, to the ataxia-telangiectasia mutated (ATM) gene. To understand how this binding affects the alteration of ATM expression and to identify biomarkers for the detection of esophageal cancer, we analyzed ATM mRNA expression in tissue specimens from patients with esophageal SCC and premalignant lesions using in situ hybridization. We then performed in vitro experiments to verify and extend our ex vivo observations. We found that ATM expression was increased in esophageal SCC and its premalignant lesions when compared with normal tissues and that increased ATM expression was associated with tobacco smoke exposure and tumor de-differentiation. Moreover, BPDE induced ATM expression in esophageal SCC cell lines in a time-dependent manner. In summary, the BPDE in tobacco smoke may be responsible for increased ATM expression in premalignant and malignant esophageal tissues. Our findings suggest that the ATM gene should be further evaluated as a biomarker for the early detection of esophageal cancer and tobacco use in patients.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro.

Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast-like cells (MC3T3-E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 x 10(5) Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37 degrees C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3-E1 cells were cultured in alpha-minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2 incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2 (PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose-dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2, which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.

Animals↗