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M Tada

Publications and source records attributed to M Tada.

At least 199 records · Page 11Linked to original sources
PubMed1997

Reappraisal of p53 mutations in human malignant astrocytic neoplasms by p53 functional assay: comparison with conventional structural analyses.

We previously reported clonal expansion of p53 mutations in malignant astrocytic tumors detected with a yeast p53 functional assay that measures mutant p53 alleles quantitatively and loss of p53 transcriptional competence qualitatively (Tada et al., Int J Cancer 67:447-450, 1996). This method selectively detects inactivating mutations and is relatively insensitive to contamination of tumor samples with normal tissue. To determine whether the mutation frequency and spectrum detected in this way differ from those seen with conventional techniques, 54 malignant astrocytomas were tested with the yeast assay, and the abnormalities detected were characterized by DNA sequencing. Inactivating p53 mutations were found in 67% of anaplastic astrocytomas and 41% of glioblastomas. Overall, mutations were found in 48% of tumors, compared with only 29% in previous studies (P < 0.005), a difference that probably reflects the greater sensitivity of the yeast assay than of conventional techniques. The frequency of mutations in anaplastic astrocytomas (in our study plus published studies) was significantly higher than in glioblastomas (39% vs 29%; P < 0.05). This suggests that acquisition of p53 mutations is not rate limiting for progression to glioblastoma and that many glioblastomas develop by p53-independent pathways. Sequencing of mutant p53 cDNAs rescued from yeast showed that the mutation spectrum for functionally inactive mutants was nearly identical to the spectra from previous studies on structural mutants, indicating that transcriptional activity is the critical biological target of p53 mutation in malignant astrocytomas.

Adult↗
PubMed1997

Effects of preconditioning with ebselen on glutathione metabolism and stress protein expression.

Selenium induces several proteins, including glutathione and stress proteins. These proteins have been shown to be cardioprotective against oxidative injury. To determine whether ebselen, a seleno-organic compound, can also induce these proteins and exert cardioprotective action, we examined the effects of preconditioning with ebselen on glutathione metabolism and stress protein expression and on myocyte injury induced by oxidative stress. Treatment of cultured cardiac myocytes with ebselen (0.3-30 microM) for 24 hr increased the reduced glutathione content. Glutathione reductase activity, but not glutathione peroxidase activity, was significantly elevated in a dose-dependent manner. Pretreatment with ebselen increased the expression of such stress proteins as heat shock protein 70 and heme oxygenase-1 (heat shock protein 32) in cardiac myocytes, as assessed by Western blotting. Expression of heat shock protein 70 was increased only at a higher dose of ebselen (30 microM), whereas expression of heme oxygenase-1 was markedly increased at a lower dose of ebselen (3 microM). Under these conditions, the myocyte injury induced by hydrogen peroxide or simulated ischemia/reperfusion, assessed by the release of lactate dehydrogenase into the culture medium, was reduced by ebselen pretreatment in a dose-dependent manner. Results indicated that cardiac myocytes pharmacologically preconditioned with ebselen for 24 hr exhibited resistance to oxidative injury, possibly via the up-regulation of glutathione metabolism and the expression of stress proteins.

Animals↗
PubMed1997

[A case report of Vp3 hepatocellular carcinoma responding to multidisciplinary treatment].

We report a 60-year-old man who had hepatocellular carcinoma with tumor thrombus in the main portal vein and left portal branch (VP3HCC). He was treated with transarterial chemo-embolization, surgical resection and intra-arterial infusion chemotherapy. He is now surviving without any sign of recurrence for 12 months after the initial therapy, even though the prognosis of VP3HCC is poor. This is a case in which the effect of multidisciplinary treatment was indicated.

Antibiotics, Antineoplastic↗
PubMed1997

Inhibitory effect of lead on tube formation by cultured human vascular endothelial cells.

The effect of lead acetate (Pb) on the formation of capillary-like structures (tube formation) by cultured human umbilical vascular endothelial cells (HUVECs) was examined. HUVECs were seeded on a gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.3-30.0 microM Pb for 24 hours inhibited the tube formation dose-dependently. The length of tube formation decreased time-dependently with 3.0-10.0 microM Pb. To elucidate the main target factor of Pb for this inhibition, the effects of Pb on the activity of protein kinase C (PKC) and Matrigel were examined. The addition of beta-phorbol 12-myristate 13-acetate (PMA, 50 nM), an activator of PKC, and isoquinolinesulfonamide derivative (H-7, 30 microM), an inhibitor of PKC, showed an increase and decrease in the tube formation, respectively. However, the results of simultaneous addition of Pb and either PMA or H-7 to HUVECs indicated that PMA and H-7 acted not synergistically but additively. When PKC activities in HUVECs were measured by a colorimetric assay after treatments with 3.0-10.0 microM Pb for 24 hours, there was no significant change in PKC activity in the cells. The Pb-inhibition of tube formation was suggested to be independent of PKC activity. Pretreatment of Matrigel with 3.0-10.0 microM Pb for different periods decreased the tube formation dose- and time-dependently. These findings suggest that Pb can inhibit the tube formation by HUVECs dose- and time-dependently and that the inhibitory effect of Pb could be dependent on the degeneration of Matrigel, not on PKC activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Receptor protein tyrosine kinase DDR is up-regulated by p53 protein.

We have previously reported on radiation-induction of ptk-3 in rat astrocyte culture [Sakuma et al. (1995) Radiat. Res. 143, 1-7]. Ptk-3 was considered to be a rat version of human DDR (discoidin domain receptor). We cloned and analyzed genomic DNA of the DDR and its promoter region. We discovered that the promoter region contained a consensus sequence of the p53 tumor suppressor binding site. Adenovirus-mediated p53 transfection induced a high level of DDR mRNA in SAOS2 human osteosarcoma cells. These results indicate that DDR is up-regulated by the p53 protein.

Blotting, Northern↗
PubMed1996

Phospholamban regulates the Ca2+-ATPase through intramembrane interactions.

There is clear evidence for direct regulatory protein-protein interactions between phospholamban (PLN) and the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) in cytoplasmic domains, but there is less clear evidence for regulatory interactions in the transmembrane domains of the two proteins. We have now coexpressed SERCA isoforms with the transmembrane sequence of PLN and with epitope-tagged transmembrane sequences of PLN to study intramembrane interactions in the absence of cytoplasmic interactions. Coexpression of the transmembrane sequence of phospholamban (Met-PLN28-52) with SERCA1a, SERCA2a, and SERCA3 inhibited Ca2+ transport by lowering apparent Ca2+ affinity. Addition of the hemagglutinin (HA) epitope to the transmembrane sequence of PLN (HA-PLN28-52) or deletion of PLN residues 21-29 (PLN1-20-PLN30-52) "supershifted" apparent Ca2+ affinity to values lower than those observed with native PLN without uncoupling Ca2+ transport from ATP hydrolysis. Inhibition by PLN1-20-PLN30-52 or by Flag-PLN28-52 was reversed by PLN antibody or by Flag antibody, demonstrating that inhibition by these constructs is reversible and that the inhibitory constructs are properly oriented in the membrane. These results suggest that PLN modulates the apparent Ca2+ affinity of SERCA2a through intramembrane interactions, which are disrupted at long range and in concert with disruption of the well characterized cytoplasmic interactions.

Adenosine Triphosphate↗
PubMed1996

Cloning and characterization of the 5'-upstream regulatory region of the Ca(2+)-release channel gene of cardiac sarcoplasmic reticulum.

To elucidate the transcriptional regulation mechanism for the Ca(2+)-release channel gene of the cardiac sarcoplasmic reticulum (RYR2), we isolated and analyzed the 5'-upstream flanking region of the gene. Sequence analysis indicated that the core promoter region lacks canonical TATA and CAAT boxes, but contains three overlapping GC boxes. A gel shift assay indicated that Sp1 binds to the region containing the GC boxes. Different 5'-deletion constructs in the 5'-flanking region of the RYR2 gene were fused to the luciferase gene, and their promoter activity in rat neonatal cardiac myocytes was subsequently determined. The results revealed the presence of a region containing positive regulatory elements in the 5'-flanking region. Analyses of substitutional mutations introduced into the GC boxes and the regulatory region indicated that in addition to the GC box located at -56 to -51, two regulatory elements (RYR2P1 and RYR2P2) are essential for the promoter activity. These results indicated that Sp1 and transcription factors that bind to RYR2P1 and RYR2P2 cooperatively enhance the expression of the RYR2 gene. In a transient transfection experiment involving promoter-luciferase gene constructs in skeletal muscle cells, we identified a negative regulatory region between positions -209 and -90 that represses the expression of the RYR2 gene in skeletal muscle cells.

Animals↗
PubMed1996

Clonality and stability of the p53 gene in human astrocytic tumor cells: quantitative analysis of p53 gene mutations by yeast functional assay.

Mutation of the p53 gene is found in about one third of astrocytic brain tumors, and expansion of tumor cell clones containing mutant p53 has been implicated in astrocytic tumor progression. However, admixture of normal cells in astrocytic tumor specimens limits the power of traditional studies of tumor cell clonality. To address this problem we have employed a yeast p53 functional assay that scores the content of mutant p53 alleles in tumors and cell lines quantitatively. We have analyzed 17 cases where matching tumor material and derived cell lines were available. The yeast assay gave > 20% red (i.e., mutant p53-containing) yeast colonies in 7 out of 17 cases. One case had no mutations in the primary tumor but gave 76% red colonies in a recurrence, clearly demonstrating tumor overgrowth by a mutant clone. During early passages of cultured tumor cells, mutant p53 content increased rapidly with passage due to outgrowth of mutant clones from a heterogeneous starting population. In addition, de novo p53 mutations appeared during culture in 2 cases. This indicates that there is stronger selective pressure for mutation during the establishment of cell lines in vitro than during tumor growth in vivo. Our results demonstrate the utility of the p53 functional assay for studies of clonality and support the hypothesis of clonal progression of brain tumors in vivo.

Astrocytoma↗
PubMed1996

Activation of the inactive X chromosome induced by cell fusion between a murine EC and female somatic cell accompanies reproducible changes in the methylation pattern of the Xist gene.

Mouse embryonal carcinoma (EC) cell lines are divided into two classes with or without the capability of reactivating the inactive X chromosome from a fusion partner of female lymphocyte. The 5' region of Xist was partially methylated in reactivating-competent EC cells but was fully methylated in reactivating-incompetent EC cells having a single X chromosome. Partial or heterogeneous methylation implies methylation of each CpG site in about half of the cell independently of methylation status of neighboring CpG sites. Fusion of the reactivating-competent EC cells with female lymphocytes induced not only de novo methylation in the 5' region of Xist allele on the hitherto inactivated X chromosome, but also demethylation of the same region of Xist on the other X chromosome from the female somatic cell. In contrast, no such changes occurred in hybrid cells involving reactivating-incompetent EC cells. Thus, partial methylation of the 5' region of Xist most probably maintained by low maintenance and high de novo methylation efficiency is correlated with reactivation potential of the EC cell. It is possible that this unique methylation pattern is implicated in random X inactivation in EC-hybrid cells in vitro and in epiblast cells in vivo.

Animals↗
PubMed1996

Amelioration of severity of myocardial injury by a nitric oxide donor in rabbits fed a cholesterol-rich diet.

OBJECTIVES: This study compared the effect of a nitric oxide donor on limiting the size of infarct resulting from myocardial ischemia-reperfusion between atherosclerotic and nonatherosclerotic models. BACKGROUND: Endothelial-derived relaxation in coronary arteries affected by ischemia is substantially impaired after reperfusion, and this impairment may exacerbate the myocardial ischemia-reperfusion injury. In animals with experimental atherosclerosis, release of endothelial-derived relaxing factor is also decreased, and the propagation of myocardial infarction could be exacerbated. METHODS: We examined the extent of myocardial injury induced by ischemia (30 min) and reperfusion (48 hr) in rabbits fed a cholesterol-rich (1%) or normal diet for 10 weeks. We also evaluated the effect of a nitric oxide donor (S-nitroso-N-acetylpenicillamine [SNAP], a nitric oxide precursor (L-arginine) or a degradation product of SNAP (N-acetylpenicillamine) on infarct size in these models. RESULTS: Severity of myocardial injury was significantly exacerbated in cholesterol-fed rabbits (75.2 +/- 4.4% [mean +/- SEM]) compared with that in non-cholesterol-fed rabbits (53.2 +/- 5.2%). This exacerbation was prevented by treatment with SNAP (50.2 +/- 6.4%) but not with L-arginine (70.5 +/- 6.0%) or N-acetylpenicillamine (70.4 +/- 4.8%) in cholesterol-fed-rabbits. However, SNAP did not limit infarct size in non-cholesterol-fed rabbits (60.8 +/- 4.2%). The rate-pressure product was similar during the course of the experiment in all the groups. CONCLUSIONS: Myocardial damage induced by ischemia-reperfusion was significantly exacerbated in rabbits fed a long-term cholesterol-rich diet but was effectively reversed by treatment with a nitric oxide donor. However, this agent did not limit infarct size in normal rabbits. Thus, a nitric oxide donor reduces myocardial infarct size in atherosclerotic but not in nonatherosclerotic rabbits.

Analysis of Variance↗
PubMed1996

Increased release of NO during ischemia reduces myocardial contractility and improves metabolic dysfunction.

BACKGROUND: We have reported that myocardial ischemia increases nitric oxide (NO) production. Several lines of evidence suggest that NO reduces myocardial contraction. Therefore, we tested whether endogenous NO decreases the inotropic response of the ischemic myocardium and whether endogenous NO is beneficial in the metabolic function of ischemic myocardium. METHODS AND RESULTS: The left anterior descending coronary artery was perfused with blood from the left carotid artery in 72 dogs. An infusion of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, did not affect fractional shortening (FS) under nonischemic conditions. After reduction of perfusion pressure so that coronary blood flow decreased to 60% of the control value, FS of the perfused area decreased, and intravenous infusion of isoproterenol increased FS. Before and during intravenous infusion of isoproterenol under conditions of coronary hypoperfusion, FS was significantly increased in the L-NAME group compared with the untreated group. Both lactate extraction ratio and the pH in coronary venous blood were significantly lower in the L-NAME-treated group than in the untreated group during coronary hypoperfusion. Infusion of L-arginine prevented the effects of L-NAME in the ischemic myocardium. CONCLUSIONS: These results indicate that endogenous NO reduces myocardial contractile function and improves myocardial metabolic function in the ischemic heart. The myocardial energy-sparing effect as well as coronary vasodilation due to NO may be beneficial to the ischemic myocardium.

Animals↗
PubMed1996

Immunohistochemical demonstration of 3,2'-dimethyl-4-aminobiphenyl DNA adduct formation in various organs of Syrian golden hamsters.

3,2'-Dimethyl-4-aminobiphenyl (DMAB), known to be a wide-spectrum genotoxic carcinogen in rats, is also tumorigenic to hamster organs such as the forestomach, small and large intestines, gallbladder and urinary bladder. Using a specific antibody against DMAB-DNA adducts, adduct formation in various organs of Syrian golden hamsters after single s.c., i.g. or i.p. injections of DMAB was immunohistochemically examined in relation to its carcinogenic specificity. The nuclei of basal cells of forestomach, epithelia of small intestine, large intestine, gallbladder and urinary bladder of hamsters were stained to various degrees. However, no differences in the formation of DMAB-DNA adducts were observed between these and non-target organs regarding carcinogenicity. The staining intensity after i.g. or i.p. injections was slightly stronger than after s.c. injection. In line with previous findings for rats, the present results indicate that adduct formation is necessary but not itself sufficient for tumor induction in the Syrian golden hamster.

Aminobiphenyl Compounds↗
PubMed1996

Heme oxygenase-1 expression and its relation to oxidative stress during primary culture of cardiomyocytes.

The inducible form of heme oxygenase (heme oxygenase-1) is a heat shock protein 32 (HSP32) whose expression is induced by numerous agents, including heme compounds and heavy metals, and during oxidative stress. The purpose of this study was to examine whether heme oxygenase-1 is induced during primary cell culture of cardiomyocytes and the relation of heme oxygenase-1 expression to oxidative stress levels. Western blot analysis and reverse transcription-polymerase chain reaction analysis showed heme oxygenase-1 expression 12-48 h after isolation of rat neonatal cardiomyocyte for culture. Its expression was barely detected immediately after isolation. Actinomycin D or cycloheximide completely suppressed such expression. Myocardial cells were exposed to oxidative stress during the first 12 h after isolation as assessed by their glutathione redox state; the ratio of reduced glutathione/oxidized glutathione was less than 10. The expression of heme oxygenase-1 was significantly reduced by treatment with reduced glutathione (58% reduction, P < 0.05), but markedly increased by treatment with hydrogen peroxide (65% increase, P < 0.05) 12 h after isolation. Expression of heat shock protein 70 was not significantly changed during primary culture incubation. Results indicate that heme oxygenase-1 is expressed during primary culture of cardiomyocytes. Its expression is closely related to the oxidative stress level of the cultured cells.

Animals↗