Non-specific detection of the centrosomes by antibodies recognizing phosphorylated ATM at serine 1981.
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Antibodies↗
Biomedical subjects
Keiji Suzuki
Publications and source records attributed to Keiji Suzuki.
Delayed activation of DNA damage checkpoint and radiation-induced genomic instability.
Ionizing radiation induces genomic instability, transmitted over many generations through the progeny of surviving cells. It is manifested as the expression of delayed effects such as delayed cell death, delayed chromosomal instability and delayed mutagenesis. Induced genomic instability exerts its delayed effects for prolonged periods of time, suggesting the presence of a mechanism by which the initial DNA damage in the surviving cells is memorized. Our recent studies have shown that transmitted memory causes delayed DNA breakage, which in turn activates DNA damage checkpoint, and is involved in delayed manifestation of genomic instability. Although the mechanism(s) involved in DNA damage memory remain to be determined, we suggest that ionizing radiation-induced mega-base deletion destabilizes chromatin structure, which can be transmitted many generations through the progeny, and is involved in initiation and perpetuation of genomic instability. The possible involvement of delayed activation of a DNA damage checkpoint in the delayed induction of genomic instability in bystander cells is also discussed.
Animals↗
Effects of antenatal corticosteroid treatment on pulmonary ventilation and circulation in neonatal lambs with hypoplastic lungs.
Our aim was to determine whether antenatal corticosteroids improve perinatal adaptation of the pulmonary circulation in lambs with lung hypoplasia (LH). LH was induced in 12 ovine fetuses between 105 and 140 days gestation (term approximately 147 days); in 6 of these the ewe was given a single dose of betamethasone (11.4 mg im) 24 hr before delivery (LH + B). All lambs, including a control group (n = 6), were delivered at approximately 140 days and ventilated for 2 hr during which arterial pressures, pulmonary blood flow (PBF), and ventilating pressure and flow were recorded. During ventilation, respiratory system compliance was lower in both LH + B and LH groups than in controls. Pulmonary vascular resistance (PVR) was lower in LH + B lambs than in LH lambs and similar to controls; PBF was reduced in LH lambs but was restored to control levels by betamethasone. The mean density of small arteries of LH + B lambs was similar to that of LH lambs (P = 0.06) and lower than in controls; the thickness of the media of small pulmonary arteries from LH + B lambs was similar to that in LH lambs and thicker than in controls. VEGF mRNA levels were not different between groups. PDGF mRNA levels in LH + B lambs were higher than in LH lambs; a similar trend (P = 0.06) was seen for PECAM-1. SP-C mRNA levels were greater in both LH and LH + B lambs than in controls. Effects of betamethasone were greater on indices of pulmonary circulation than ventilation. We conclude that a single dose of maternal betamethasone 24 hr prior to birth has significant favorable effects on the postnatal adaptation of the pulmonary circulation in lambs with LH.
Animals↗
Reconstruction using bone lengthening of the residual digit after amputation for the treatment of digital malignant tumors.
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Adult↗
Chronic endotoxin exposure causes brain injury in the ovine fetus in the absence of hypoxemia.
OBJECTIVE: Intrauterine infection has been linked to brain injury in human infants, although the mechanisms are not fully understood. We recently showed that repeated acute exposure of preterm fetal sheep to bacterial endotoxin (lipopolysaccharide [LPS]) results in fetal hypoxemia, hypotension, increased systemic proinflammatory cytokines, and brain damage, including white matter injury. However, it is not clear whether this injury is caused by reduced cerebral oxygen delivery or inflammatory pathways independent of hypoxia. The aim of the present study was to determine the effects on the fetal brain and placenta of a chronic intrauterine inflammatory state, induced by LPS infusion into the fetal circulation, a model that did not cause hypoxia. METHODS: At 0.65 of term, eight catheterized fetal sheep received intravenous infusions of LPS (5 to 15 mug) over 5 days; control fetuses received saline. Fetal physiologic responses were monitored throughout the infusion. Fetal brain and placental tissues were examined histologically 6 days after the conclusion of the infusion. RESULTS: LPS infusions did not result in physiologically significant alterations to fetal blood gases or mean arterial pressure; however, plasma proinflammatory cytokine levels were elevated. Following LPS exposure there was no difference in fetal body or brain weights (P >.05); placental weight was reduced (P <.05), consistent with reduced placentome cross-sectional area (P <.05). In the cerebral hemispheres subcortical white matter injury was present in six LPS-exposed fetuses and included axonal damage, microgliosis, oligodendrocyte injury, and increased beta amyloid precursor protein (beta-APP) expression. CONCLUSIONS: Chronic, systemic exposure of the fetus to LPS resulted in fetal brain damage in the absence of hypoxemia or hypotension, although the resulting injury was less severe than following repeated acute exposure.
Amyloid beta-Protein Precursor↗
Clinical validity of proliferating cell nuclear antigen as an objective marker for evaluating biologic features in patients with untreated prostate cancer.
BACKGROUND: Although Gleason grading may be the most useful system for evaluating biological activity of untreated prostate cancer, lack of interobserver validity with Gleason scores (GS) is an unsolved issue. In this study, the proliferating cell nuclear antigen labeling index (PCNA LI) in untreated prostate cancer was investigated in order to clarify the usefulness of supplemental and objective markers for evaluating the biologic features of prostate cancer. METHODS: Sixty cases of prostate cancer were randomly selected from the cancer registry in Gunma University Hospital for this study. PCNA LI were evaluated using paraffin-embedded biopsy cores taken at diagnosis. Correlation of PCNA LI with the Gleason grading system, clinical stage, serum prostate-specific antigen (PSA) levels and age were evaluated. Cumulative rates of freedom from cause-specific death were also evaluated stratified by various clinicopathologic features, including PCNA LI using Kaplan-Meier analysis. RESULTS: Proliferating cell nuclear antigen labeling index was significantly higher in patients with PSA levels over 100 ng/mL, advanced clinical stage (>T4, N1 or M1 disease), higher Gleason grade or with a higher GS than in those with other clinicopathologic features. The 5-year cumulative rate of death from prostate cancer was significantly higher at 62% in patients with a PCNA LI of 20 or more than those with PCNA LI of less than 20, who accounted for 4%. CONCLUSIONS: Proliferating cell nuclear antigen labeling index in combination with Gleason grading system may be of clinical value in evaluating biologic features and also in predicting cause specific survival of prostate cancer in an objective, reliable and reproducible manner.
Aged↗
[Low-dose radiation effects and intracellular signaling pathways].
Accumulated evidence has shown that exposure to low-dose radiation, especially doses less than 0.1 Gy, induces observable effects on mammalian cells. However, the underlying molecular mechanisms have not yet been clarified. Recently, it has been shown that low-dose radiation stimulates growth factor receptor, which results in a sequential activation of the mitogen-activated protein kinase pathway. In addition to the activation of the membrane-bound pathways, it is becoming evident that nuclear pathways are also activated by low-dose radiation. Ionizing radiation has detrimental effects on chromatin structure, since radiation-induced DNA double-strand breaks result in discontinuity of nucleosomes. Recently, it has been shown that ATM protein, the product of the ATM gene mutated in ataxia-telangiectasia, recognizes alteration in the chromatin structure, and it is activated through intermolecular autophosphorylation at serine 1981. Using antibodies against phosphorylated ATM, we found that the activated and phosphorylated ATM protein is detected as discrete foci in the nucleus between doses of 10 mGy and 1 Gy. Interestingly, the size of the foci induced by low-dose radiation was equivalent to the foci induced by high-dose radiation. These results indicate that the initial signal is amplified through foci growth, and cells evolve a system by which they can respond to a small number of DNA double-strand breaks. From these results, it can be concluded that low-dose radiation is sensed both in the membrane and in the nucleus, and activation of multiple signal transduction pathways could be involved in manifestations of low-dose effects.
Ataxia Telangiectasia↗
Phosphorylated histone H2AX foci persist on rejoined mitotic chromosomes in normal human diploid cells exposed to ionizing radiation.
Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that "residual" foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.
Ataxia Telangiectasia Mutated Proteins↗
Qualitative and quantitative analysis of phosphorylated ATM foci induced by low-dose ionizing radiation.
We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose.
Ataxia Telangiectasia Mutated Proteins↗
Histone H2AX phosphorylation in normal human cells irradiated with focused ultrasoft X rays: evidence for chromatin movement during repair.
DNA repair within the cell nucleus is a dynamic process involving a close interaction between repair proteins and chromatin structure. Recent studies have indicated a quantitative relationship between DNA double-strand break induction and histone H2AX phosphorylation. The dynamics of this process within individual cell nuclei is unknown. To address this, we have used a novel focused ultrasoft X-ray microprobe that is capable of inducing localized DNA damage within a subnuclear area of intact cells with a 2.5-microm-diameter beam spot. The present investigation was undertaken to explore the influence of focused irradiation of individual nuclei with 1.49 keV characteristic aluminum K-shell (AlK) X rays on H2AX phosphorylation in normal human cells. Immunofluorescence analyses revealed that significant diffusion of the initial spots of clustered foci of phosphorylated H2AX occurred in a time-dependent fashion after exposure to AlK X rays. Irradiation under cooled conditions resulted in a reduction in the size of spots of clustered foci of phosphorylated H2AX as well as of individual phosphorylated H2AX foci. These findings strongly suggest that diffusion of the chromatin microenvironment occurs during the repair of DNA damage. We also found that AlK ultrasoft X rays (71 foci per gray) were 2.2-fold more effective at the initial formation of phosphorylated H2AX foci than with conventional X rays (32 foci per gray), and that the time required to eliminate 50% of the initial number of foci was 3.4-fold longer in AlK-irradiated cells than that in cells exposed to conventional X rays. For conventional X rays, we also report significant accumulation of larger-sized foci at longer times after irradiation.
Cell Line↗
Interstitial chromatin alteration causes persistent p53 activation involved in the radiation-induced senescence-like growth arrest.
Various stresses including ionizing radiation give normal human fibroblasts a phenotype of senescence-like growth arrest (SLGA), manifested by p53-dependent irreversible G1 arrest. To determine the mechanism of persistent activation of p53, we examined phosphorylated Ataxia telangiectasia mutated (ATM) and phosphorylated histone H2AX foci formation after X-irradiation. Although the multiple tiny foci, detected soon after (<30 min) irradiation, gradually disappeared, some of these foci changed to large foci and persisted for 5 days. Large foci containing phosphorylated ATM and gamma-H2AX co-localized and foci with p53 phosphorylated at serine 15 also showed the same distribution. Interestingly, the signals obtained by telomere fluorescence in situ hybridization (FISH) assay did not co-localize with 90% of the large foci. Our results indicate that chromatin alteration in interstitial chromosomal regions is the most likely cause of continuous activation of p53, which results in the induction of SLGA by ionizing radiation.
Cell Cycle↗
Abnormal stability of wild-type p53 protein in a human lung carcinoma cell line.
We report here that ectopically expressed wild-type p53 protein showed more than 6 times longer half-life than normal human fibroblasts in NCl-H1299, a widely used cell line derived from non-small cell lung carcinoma lacking the expression of p53 protein. We found no abnormality in the phosphorylation and ubiquitination of p53, and the expression levels of MDM2. Although proteasome activity measured in vitro was not significantly different between the tumor cell line and normal human fibroblasts, proteasome inhibitors, ALLN, MG115, and MG132, did not accumulate p53 protein in the tumor cell line, but did accumulate p53 in normal human cells. These results provide a novel mechanism, by which p53 is stabilized in tumor cells, and they suggest that a mediator should exist between ubiquitinated p53 and proteasome, which may be defective in H1299 cells.
Cell Line, Tumor↗
Effect of lung hypoplasia on birth-related changes in the pulmonary circulation in sheep.
Lung hypoplasia (LH) is a serious cause of neonatal compromise, but little is known of its functional effects on the pulmonary circulation. Our aim was to characterize birth-related changes in the pulmonary circulation of newborn lambs with LH and to compare them with alterations in respiratory function. LH was induced in six ovine fetuses by the creation of a tracheo-amniotic shunt as well as amniotic fluid drainage starting at 105.6+/-1.5 (mean+/-SEM) days of gestation (term approximately 147 d). At 139.9+/-0.3 d, fetuses were exteriorized under anesthesia to implant vascular catheters and an ultrasonic flow probe around the left pulmonary artery. The lambs then were delivered and ventilated for 2 h, during which systemic and pulmonary artery pressures, left pulmonary blood flow, and measures of respiratory function were recorded. At autopsy, lungs were weighed and volume was measured at 20 cm H2O. In LH lambs, lung weight was 25% lower and respiratory system compliance was 30% lower than in controls. Mean pulmonary blood flow in LH lambs was 42% lower and pulmonary vascular resistance was 138% higher than in controls. Morphometry showed that volume density of pulmonary arteries in LH was 30% lower than in controls. We conclude that, in this LH model, changes in ventilatory indices were proportional to the change in lung size, whereas changes in the pulmonary circulation were greater than the change in lung size and were associated with reduced density of pulmonary arteries. LH severely impairs normal adaptation of the pulmonary circulation in the perinatal period.
Animals↗
Pulmonary function and structure following mild preterm birth in lambs.
Our objective was to determine whether postnatal respiratory function, lung growth, and lung structure are affected by preterm birth which did not require neonatal respiratory support. Two groups of preterm (P) lambs were delivered 2 weeks before term, at 133 days of gestational age (GA). Tissue was collected at term equivalent age (TEA, 147 days GA) in one P group and at 6 weeks post-TEA in the other. Tissue was also collected from control (C) lambs soon after term birth (TEA) and at 6 weeks post-TEA. Lung function was assessed at TEA and 6 weeks post-TEA. Respiratory system compliance (Crs/kg BWT) was not different between P and C groups at TEA, but was higher (P = 0.02) in P lambs at 6 weeks post-TEA. Pulmonary resistance was 62% higher in P lambs than controls (P = 0.07) at TEA, and remained higher at 6 weeks post-TEA. Lung weights (wet and dry) were greater (P < 0.05) in preterm animals at both ages; when adjusted for body weight, only dry lung weight remained higher at 6 weeks post-TEA. Alveoli were more numerous (P = 0.05) and smaller (P = 0.05) in preterm lambs compared to controls at both ages. Alveolar septa were 33% thicker and the blood-air barrier was 26% thicker in P lambs than in controls at TEA, and remained thicker at 6 weeks post-TEA. In P lambs, the airway epithelium was thicker at TEA and 6 weeks post-TEA. At TEA, pulmonary tropoelastin expression was 27% lower in P lambs. At 6 weeks post-TEA, dry lung weight and lung protein content were approximately 50% greater in preterm lambs than in controls (P < 0.05), whereas lung DNA, elastin, and collagen contents were similar in the two groups. We conclude that mild preterm birth per se leads to both transient and persistent changes in lung development. Persistent increases in lung protein content and in the thickness of the airway epithelium, and a greater number of smaller alveolar, may alter later lung function.
Animals↗
Interferon-alpha-induced changes in metallothionein expression in liver biopsies from patients with chronic hepatitis C.
An association between reactive oxygen species and liver damage has been postulated in the course of hepatitis C virus (HCV) infection. Metallothionein (MT), induced by HCV core protein and interferon (IFN), plays a role in scavenging free radicals. MT expression in liver biopsies obtained from 21 patients with chronic HCV infection before and after IFN-alpha therapy was investigated. Changes in Knodell histological activity index (HAI) scores, MT protein levels (immunohistochemistry), MT-I and MT-II messenger (m)RNA expression levels (in situ hybridization) and proliferating cell nuclear antigen (PCNA) labelling index were determined and compared in serial liver specimens. MT staining was clustered around the portal tracts with inflammatory cells and fibrosis. The pattern of MT protein before IFN-alpha therapy was similar in all patients, but was higher in IFN-sustained responders than in nonresponders after IFN-alpha therapy. HAI scores and PCNA labelling indexes were significantly reduced after IFN-alpha therapy. MT-II mRNA expression correlated positively with PCNA index before therapy and with HAI scores after therapy (P<0.05). No correlation was found between MT-I mRNA and HAI scores or PCNA index. The findings indicate that IFN-alpha-induced hepatic MT may participate in the therapeutic effects of IFN-alpha for HCV. In addition, MT-II mRNA expression may be involved in cell proliferation in the livers of patients with chronic HCV infection.
Antiviral Agents↗
Differences in effects of oncogenes on sensitivity to anticancer drugs.
Methods to predict the responsiveness of a particular tumor to a particular anticancer drug are desirable not only for chemotherapy but also for chemoradiotherapy. Here, we examined the effects of viral or activated oncogenes on sensitivity to anticancer drugs by using SHOK (Syrian hamster Osaka-Kanazawa) cells and their transfectants. The IC50 of each transfectant was compared with that of the pSV2Neo transfected control. Cells transfected with the c-myc, v-mos, or v-fgr gene increased their sensitivity to bleomycin, while those transfected with the H-ras gene developed resistance. Resistance to cisplatin was conferred by the introduction of the H-ras or c-cot gene. In the case of adriamycin, the c-myc or c-cot transfectant increased sensitivity and the H-ras transfectant decreased it. Mitomycin C resistance was observed by the introduction of the K-ras gene. Thus, the H-ras gene was found to be involved in the development of resistance to three of the four anticancer drugs. In addition, we have for the first time shown that mos and cot have an effect on sensitivity to three and all of the four anticancer drugs, respectively. These results suggest that the expression of each oncogene would differently affect sensitivity to the four anticancer drugs used in this study, and this property could be a possible marker to predict chemosensitivity.
Animals↗
Exogenous expression of exonuclease domain-deleted WRN interferes with the repair of radiation-induced DNA damages.
Werner syndrome (WS) is an autosomal recessive disease characterized by multiple progeroid features. The gene responsible for WS, WRN, is a member of the human RecQ helicase family. WRN is unique among this family, associated with an exonuclease activity. In the present study, we established the human 293-derived cell lines, which expressed exogenously truncated WRN protein, lacking the N-terminal exonuclease domain but having normal helicase activity, and found that they were slightly, but nonetheless significantly, radiosensitive than control cell lines, into which the empty vector had been introduced. The truncated WRN-expressing cells also exhibited increased numbers of micronuclei, chromosome aberrations, and the foci of phosphorylated histone H2AX with X-rays. These results suggested a function of WRN exonuclease activity that is separable from helicase activity and is essential for the repair of radiation-induced DNA damages.
Cell Line↗
A comparison of interobserver reproducibility of Gleason grading of prostatic carcinoma in Japan and the United States.
CONTEXT: Gleason grading is now the sole prostatic carcinoma grading system recommended by the World Health Organization. It is imperative that there be good interobserver reproducibility within this system worldwide. To our knowledge, there are no studies, using the same specimens, that compare the interobserver reproducibility of Gleason grading in Japan and the United States. OBJECTIVE: To compare the interobserver reproducibility of Gleason grading of prostatic carcinoma in Japan and the United States using, in Japan, images from the identical biopsy glass slides that were originally graded in the United States. DESIGN: Microsopic images from 37 needle biopsies of prostatic carcinoma were placed on CD-ROM and distributed to 14 Japanese pathologists for grading. These 14 physicians included 8 general pathologists and 6 pathologists with a special interest in urologic pathology. The needle biopsies had been previously reviewed so that a consensus diagnosis could be formed by a panel of urologic pathologists in the United States and Canada. Interobserver agreement with the consensus diagnoses was calculated by determining the overall kappa coefficient for the Japanese pathologists and then compared to the interobserver agreement among American general pathologists who had previously graded identical needle biopsies from which the CD-ROM images had been taken. RESULTS: The interobserver agreement with the consensus diagnoses for the 4 Gleason grading groups (Gleason grades 2-4, 5-6, 7, and 8-10) among the Japanese urologic pathologists in this series of cases was substantial (overall kappa = 0.68), and for the Japanese general pathologists, it was moderate (overall kappa = 0.49), similar to that reported in the earlier study of American general pathologists (overall kappa = 0.44). The major interobserver reproducibility problem for both Japanese and American general pathologists is undergrading. The major areas of undergrading are the underdiagnosis of Gleason scores 5-6 as Gleason scores 2-4, and the underdiagnosis of cribriform sheets and fragments of cribriform Gleason pattern 4 carcinoma as Gleason pattern 3. CONCLUSIONS: The interobserver reproducibility of the Gleason grading for this collection of specimens was similar among Japanese and American general pathologists. The overall kappa values for these generalists of 0.44 and 0.49 are only in the moderate (0.41-0.60) range of interobserver agreement when compared to 0.68, substantial (0.61-0.80) agreement, for Japanese urologic pathologists. Educational efforts to improve Gleason grading have been shown to be effective and are clearly warranted.
Adenocarcinoma↗