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Biomedical subjects

H Fujiki

Publications and source records attributed to H Fujiki.

At least 19 recordsLinked to original sources
PubMed1992

Differentiation induction in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A.

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.

Antibodies, Monoclonal↗
PubMed1992

Cytoskeletal integrity in interphase cells requires protein phosphatase activity.

Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.

Animals↗
PubMed1992

Inhibition of protein phosphatases blocks myogenesis by first altering MyoD binding activity.

To examine the role of protein phosphatases in skeletal muscle differentiation, C2C12 myoblasts were treated with okadaic acid, a potent in vitro inhibitor of protein phosphatases 1 and 2A which regulate various cellular events in intact cells. We now show that okadaic acid treatment of the mouse myoblast C2C12 cell line reversibly altered the morphology of the cells and blocked differentiation. At a molecular level, it extinguished expression of the myogenic determination genes, MyoD1 and myogenin, but induced the expression of an inhibitor of differentiation, Id. Analysis of the MyoD1 promoter showed that inhibition of MyoD1 expression by okadaic acid occurs at the transcriptional level. These changes occur 10-20 h after okadaic acid treatment. However, within 1 h of treatment the ability of muscle extracts to support a specific MyoD-dependent gel mobility shift using a MyoD DNA binding site is lost. These data suggest that protein phosphatases play an important role during myogenic differentiation.

Animals↗
PubMed1992

Stimulation of arachidonic acid metabolism by a streptococcal preparation (OK-432) in rat peritoneal macrophages.

A streptococcal preparation OK-432 is reported to be an immunopotentiator and a potent antitumor agent. In order to elucidate the mechanism of biologic action, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages were investigated. Prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were found to be stimulated by OK-432 in a concentration-dependent manner (5 to 80 micrograms/ml). Heat-treatment of OK-432 further stimulated its effects. These stimulative effects on arachidonic acid metabolism by OK-432 were not observed in MDCK cells that have no phagocytotic activity. Furthermore, cytochalasin B treatment completely suppressed the stimulative effects induced by OK-432 in macrophages. These results strongly indicate that the stimulative effects by OK-432 on arachidonic acid metabolism are dependent on phagocytosis of OK-432 particles. Significance of stimulation of arachidonic acid metabolism in macrophages by OK-432 for its biological effects is discussed.

Animals↗
PubMed1992

Increased phosphorylation of nuclear phosphoproteins in human lung-cancer cells resistant to cis-diamminedichloroplatinum (II).

A novel non-phorbol-ester-like tumor promoter, okadaic acid (OA) has been shown to be an inhibitor of protein phosphatase I and IIA and, thus, to cause an "apparent activation" of protein kinase C (PKC). We previously showed that cis-diamminedichloroplatinum(II) (CDDP)-resistant cells, PC-9/CDDP, were cross-resistant to OA and that the cross-resistance was not due to the increased efflux of OA. We hypothesized that the phosphorylation status of some cellular proteins might be important in CDDP-resistance. No significant difference in PKC activity or total protein phosphatase activity measured in vitro was seen between PC-9 and PC-9/CDDP cells, nor in their sensitivity to inhibition by OA, nor in the amount of phosphorylation of whole cells or TCA-insoluble material. By SDS-PAGE after incubation of intact cells with 32P, we detected a marked increase, compared to PC-9 cells, in phosphorylation of the nuclear proteins of MW 32 and 20 kDa in CDDP-resistant PC-9/CDDP cells with no apparent difference in protein content. When phosphorylation of nuclear proteins observed in PC-9/CDDP cells was analyzed by 2-dimensional SDS-PAGE, the 32-kDa protein had a PI of about 4.5. The 32-kDa and 20-kDa bands were increased in a dose-dependent manner by CDDP treatment. On the other hand, no increase in phosphorylation of these proteins was observed in parental PC-9 cells. These results demonstrate a marked difference in the phosphorylation status of specific nuclear proteins between parental and CDDP-resistant cell lines, which may be related to CDDP-resistance.

Adenocarcinoma↗
PubMed1992

Okadaic acid induces the expression of both early and secondary response genes in mouse keratinocytes.

Okadaic acid (OA), a potent mouse skin tumor promoter and inhibitor of the protein phosphatases 1 and 2A, was investigated for its effects on the expression of tumor-associated early and secondary response genes in mouse keratinocytes. Adult mice were treated topically with 12.5 nmol of OA, and the steady-state levels of various gene transcripts in the skin were determined at different times after treatment. The nuclear proto-oncogenes c-fos and c-jun are referred to as early response genes because the classical tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces their expression to maximal levels within 2 h after treatment. OA induced the expression of c-fos 2-72 h after treatment, with two peaks at 6 and 48 h. The steady-state level of expression of c-jun was relatively high in untreated skin, and OA induced a slight increase in its expression from 12 to 48 h after treatment. Transin and plasminogen-activator (PA) urokinase, whose induced expression peaks at least 4 h after TPA treatment, are referred to as secondary response genes. OA induced their expression more slowly than TPA. In mouse papilloma cell line 308, OA induced higher and more sustained steady-state levels of c-jun and c-fos than an equimolar dose of TPA. Transin and PA-urokinase were induced to similar levels by TPA and OA in 308 cells; however, the induction of these genes by OA was slower than induction by TPA. The existence of different patterns of induced expression of early and secondary response genes by OA and TPA suggests that these tumor promoters affect gene expression in mouse keratinocytes through different pathways.

Animals↗
PubMed1992

Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR.

Certain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the two L-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathione S-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.

Alanine Transaminase↗
PubMed1992

Cardiovascular actions of OPC-18790: a novel positive inotropic agent with little chronotropic action.

OPC-18790 [(+/-)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]- 2(1H)-quinolinone], a novel positive inotropic agent, was investigated in several in vitro and in vivo experiments to elucidate its cardiovascular effects and its mechanism of action. In isolated blood-perfused dog heart preparations, OPC-18790 increased contractile force at 10 to 1,000 nmol i.a.; increased coronary arterial blood flow at 30 to 1,000 nmol; and decreased sinus rate slightly at 1,000 nmol. Atrio-ventricular nodal conduction was slightly facilitated with OPC-18790 (10 to 1,000 nmol), whereas ventricular automaticity tended to decrease. OPC-18790 (10(-6) to 10(-4) M) increased contractile force in isolated ventricular muscles of dogs, cats, rabbits and guinea pigs but not rats. OPC-18790 increased left ventricular contractile force dose-dependently in anesthetized open-chest dogs and in conscious dogs with slight or no changes in heart rate and blood pressure. The positive inotropic effect of OPC-18790 was not affected by beta-blockade. OPC-18790 (10(-5) to 10(-4) M) prolonged the duration of action potential in guinea pig papillary muscles. Na+, K(+)-ATPase was not inhibited, but peak-III phosphodiesterase (low Km cyclic AMP specific fraction, inhibited by cyclic GMP) was inhibited by OPC-18790 (IC50 = 0.41 x 10(-6) M) in dog myocardium. However, such an inhibitory action of phosphodiesterase can hardly be reconciled with the lack of a positive chronotropic effect shown by OPC-18790. Thus, these results suggest that OPC-18790 may have an additional mechanism. The cardiovascular effects revealed by this study suggest that OPC-18790 may exert a beneficial effect in the treatment of congestive heart failure.

Animals↗
PubMed1992

Structurally different members of the okadaic acid class selectively inhibit protein serine/threonine but not tyrosine phosphatase activity.

The relative potencies of four main types of okadaic acid class compounds as inhibitors of the catalytic subunits of protein serine/threonine phosphatases 1 and 2A and the protein tyrosine phosphatase 1 were determined. These four types of compounds are okadaic acid, calyculin A, microcystin-LR, and tautomycin, which are isolated from different natural sources, a black sponge Halichondria okadai, a marine sponge Discodermia calyx, a blue-green alga Microcystis aeruginosa, and Streptomyces spirover ticillatus, respectively. While okadaic acid was a more effective inhibitor of protein phosphatase 2A (IC50, 0.07 nM) than protein phosphatase 1 (IC50, 3.4 nM), other compounds of the okadaic acid class were equally effective against the two protein serine/threonine phosphatases. The order of potency was microcystin greater than calyculin A greater than tautomycin, and the IC50S ranged from 0.1 to 0.7 nM. None of the okadaic acid class compounds inhibited protein tyrosine phosphatase 1 activity at concentrations up to 0.01 mM. These results indicate that the compounds of the okadaic acid class are selective inhibitors of protein serine/threonine but not tyrosine phosphatases.

Animals↗
PubMed1992

Anticarcinogenic effects of (-)-epigallocatechin gallate.

BACKGROUND: Our research objective is to develop nontoxic cancer chemopreventive agents and to apply these agents in treating humans. We are identifying agents that inhibit the process of tumor promotion in two-stage carcinogenesis experiments on mouse skin. METHODS: We review (a) the inhibitory effect of penta-O-galloyl-beta-D-glucose (5GG) on tumor promotion by teleocidin, one of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters (5GG is structurally similar to (-)-epigallocatechin gallate (EGCG) and is isolated from hydrolyzed tannic acid); (b) the inhibitory effects of EGCG, the main constituent of Japanese green tea, on tumor promotion with two tumor promoters, teleocidin and okadaic acid, a non-TPA-type tumor promoter; (c) the mechanisms of action of EGCG, a single application of which reduced the specific binding of [3H]TPA and [3H]okadaic acid to a particulate fraction of mouse skin; and (d) the anticarcinogenic effects of EGCG on duodenal carcinogenesis induced by N-ethyl-N'-nitro-N-nitrosoguanidine in male C57BL/6 mice. EGCG is a nontoxic compound. CONCLUSION: We believe that the main constituent of Japanese green tea, EGCG, is a practical cancer chemopreventive agent available in everyday life.

Animals↗
PubMed1992

An alternative theory of tissue specificity by tumor promotion of okadaic acid in glandular stomach of SD rats.

To challenge the theory of tissue specificity of tumor promoters, the biochemical and tumor promoting effects of okadaic acid (OA), a potent tumor promoter on mouse skin, were studied in the mucosa of rat glandular stomach. OA strongly inhibited protein phosphatases 1 and 2A, and increased 4-fold the phosphorylation of elongation factor 2 in vitro in the mucosa. Intubation of 10 micrograms (12.4 nmol) OA induced ornithine decarboxylase in the mucosa. Tumor promotion of OA was studied in the glandular stomach initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a two-stage carcinogenesis experiment. OA in drinking water, 10 micrograms (12.4 nmol) per rat per day from weeks 9-55 of the experiment, and 20 micrograms (24.8 nmol) from weeks 56-72, significantly enhanced development of the neoplastic changes in the glandular stomach (P < 0.05). The neoplastic changes included adenomatous hyperplasias and adenocarcinomas, both of which correspond to papillomas and carcinomas in a two-stage mouse skin carcinogenesis experiment. The percentages of neoplastic change-bearing rats of the groups treated with MNNG plus OA, MNNG alone or OA alone were 75.0, 46.4 and 0% respectively. OA enhanced tumorigenesis in the MNNG-initiated glandular stomach of rats through the same mechanisms of action as in mouse skin. The OA pathway mediated through inhibition of protein phosphatases 1 and 2A is applicable to various organs as a general mechanism of tumor promotion.

Animals↗
PubMed1992

Activity of diverse tumor promoters in a keratinocyte co-culture model of initiated epidermis.

The suppression of focal growth of initiated mouse keratinocytes by co-culture with normal keratinocytes can be overcome by treatment of co-cultures with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This keratinocyte co-culture model was developed as an analog of initiated mouse epidermis to facilitate the study of tumor promotion in cell culture. A number of promoters of TPA-type [those with protein kinase C (PKC) as receptor] were compared to non-TPA-type promoters for activity in the keratinocyte co-culture model. The TPA-type promoters teleocidin and aplysiatoxin show comparable activity to that of TPA. Exposure of co-cultures to oleoyl-2-acetylglycerol, a diacylglycerol activator of PKC, also induces focal outgrowth of initiated cells, suggesting that PKC is likely to be involved in the mechanism of action of these compounds. However, the involvement of alternative pathways (not involving PKC directly) for clonal selection are evident since the non-TPA-type promoters okadaic acid, staurosporine and thapsigargin are also active in the assay. Thus, the keratinocyte co-culture model differs from fibroblast models of normal and neoplastic co-cultures in which only TPA-type promoters are active. In further contrast to certain fibroblast co-cultures, TPA does not inhibit cell-cell communication under conditions that suppress focus formation. Taken together with previous data, we conclude that the keratinocyte co-culture model may have broad application for detecting skin tumor promoters, and may be useful for dissecting the mechanism by which normal epidermal cells suppress the growth of initiated cells.

Animals↗
PubMed1992

Hyperphosphorylation of cytokeratins 8 and 18 by microcystin-LR, a new liver tumor promoter, in primary cultured rat hepatocytes.

Microcystin-LR (MC-LR), an inhibitor of protein phosphatases 1 and 2A, is a potent tumor promoter in rat liver initiated with diethylnitrosamine. To understand its biochemical process in hepatocytes, primary cultured rat hepatocytes were treated with MC-LR. MC-LR (1 microM) induced phosphorylation of various proteins. Two 55 and 49 kDa proteins were phosphorylated at a 3-fold higher rate than other proteins, and these proteins were identified to be cytokeratins 8 and 18 respectively, by immunoprecipitation and Western blot analysis using monoclonal anti-cytokeratin 8 and 18 antibodies. The basic cytokeratins 8 and 18 showed pI 6.4 and 5.4 respectively, in two-dimensional gel electrophoresis. MC-LR dose dependently increased phosphorylation of cytokeratins 8 and 18 in a cell-free system by incubation with a cytosolic fraction of rat liver containing both protein kinases and protein phosphatases 1 and 2A, and with [gamma-32P]ATP. Cytokeratins 8 and 18 were target proteins for phosphorylation induced by inhibition of protein phosphatases 1 and 2A, in vitro and in rat hepatocytes. Thus, the treatment of rat hepatocytes with MC-LR induced hyperphosphorylation of cytokeratins 8 and 18 associated with morphological changes, indicating that intermediate filament networks were rearranged in the cytoplasm. The hyperphosphorylation of cytokeratins is a significant biochemical process associated with liver tumor promotion.

Animals↗
PubMed1992

Enhanced induction of epidermal ornithine decarboxylase activity in C57BL/6 compared to DBA/2 mice by protein kinase C-activating skin tumor promoters: relevance to genetically mediated differences in promotion susceptibility.

Previous work from our laboratory demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) or a synthetic diacylglycerol induced significantly higher epidermal ornithine decarboxylase (ODC) activity in C57BL/6 than in DBA/2 mice. To understand further the genetic basis for this strain difference, two tumor promoters were evaluated for their effects on epidermal ODC activity: teleocidin, which activates protein kinase C (PKC); and 1,8-dihydroxyl-3-methyl-9-anthrone (chrysarobin), which does not. In addition, the ODC induction response in B6D2F1 offspring and BXD recombinant inbred (RI) strains was examined following multiple treatments with TPA. A single topical application of teleocidin to mouse dorsal skin led to the hyperinduction of epidermal ODC activity in C57BL/6 mice. In contrast, while chrysarobin induced epidermal ODC activity, no significant differences in the magnitude of this response were observed in SENCAR, DBA/2 or C57BL/6 mice. Consistent with our previous findings, the magnitude of ODC induction by teleocidin in these three mouse lines (C57BL/6 greater than SENCAR greater than DBA/2) did not correlate with their susceptibility to tumor promotion by TPA (SENCAR greater than DBA/2 greater than C57BL/6). ODC activity induced by multiple application of TPA in B6DF1 mice, whose susceptibility to phorbol ester tumor promotion is inherited as an incomplete dominant trait, was comparable to that induced in C57BL/6 mice at all the doses examined. Cluster analysis of TPA-induced ODC activity in BXD RI strains allowed us tentatively to group them into four or five phenotypes and to estimate a minimum of two genetic loci controlling TPA-induced ODC activity. Furthermore, in BXD RI strains, there was no apparent relationship between the magnitude of ODC induction and responsiveness to tumor promotion or sustained hyperplasia. Collectively, these results suggest that hyperinducibility of ODC in response to PKC-activating tumor promoters is inherited as an autosomal dominant trait, and that genetic determinants for ODC induction, at least in C57BL/6 and DBA/2 mice, appear completely independent of those controlling tumor promotion susceptibility.

Animals↗
PubMed1992

Marine natural products against tumor development.

This review article deals with significant effects of marine natural products in carcinogenesis, namely as chemical probes to understand the process of carcinogenesis and as possible cancer preventive agents in humans.

Amphibian Proteins↗