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M Kasuga

Publications and source records attributed to M Kasuga.

At least 343 records · Page 19Linked to original sources
PubMed1986

[Autocrine growth factor produced by a human promyelocytic leukemia cell line].

Recently, a number of novel growth factors have been identified in culture medium conditioned by tumor cell lines. It is tempting to speculate that these growth factors produced by tumor cells could act as very good tumor markers. We have found that the conditioned medium of human promyelocytic leukemia cells (HL-60) stimulated [3H] glucose incorporation into lipids in rat adipocytes. When the conditioned medium was subjected to gel filtration on a Sephadex G-100 column, this activity was eluted in the fraction corresponding to a molecular weight of 10-12 K daltons. The production of this factor was maximal in the early log phase of cell growth and declined with increasing cell density. Differentiation of HL-60 cells to macrophages was also associated with a decrease in the production of this factor. This factor also stimulated [3H] thymidine uptake into DNA and cell proliferation in HL-60 cells themselves. These data suggest the possibility of autocrine growth of HL-60 as a result of this factor. We have subsequently tried to purify this factor. Two liters of serum-free conditioned HL-60 medium were used as a starting material. The material was purified by gel filtration, ion-exchange chromatography and high-performance liquid chromatography. Finally, 1.1 microgram of purified sample was obtained. The degree of purification was 115,613-fold. Growth factors can be very good tumor markers because this is the only tumor marker which has now been clarified to have an important role in tumor growth.

Cells, Cultured↗
PubMed1985

Variables that regulate production of insulin-like peptide(s) in human leukemia cell line (HL-60).

A human myeloid leukemia cell line (HL-60) produces a peptide or peptides with insulin-like activity which is distinct from insulin or insulin-like growth factors (somatomedins). Factors regulating the production of this peptide (HL-ILP) were explored in the present study. The production of HL-ILP was maximal in the early log phase of cell growth and declined with increasing cell density. Differentiation of HL-60 cells to macrophages, induced by dihydroxyvitamin D3 or phorbol esters, was also associated with a decrease in HL-ILP production. Glucose consumption by the cells in the early log phase was closely related with HL-ILP production, and HL-ILP was found to stimulate glucose consumption by HL-60 cells. Production of HL-ILP was dependent on glucose concentrations in the culture medium and glucose concentrations higher than 1mg/d1 suppressed the release of HL-ILP. These observations are not inconsistent with a hypothesis that HL-ILP is involved in the glucose metabolism of the HL-60 cells that produce this peptide.

Calcitriol↗
PubMed1985

Phosphorylation of tubulin and microtubule-associated proteins by the purified insulin receptor kinase.

The purified insulin receptor kinase catalyzed the phosphorylation of native tubulin and microtubule-associated proteins (MAPs; MAP2, tau) on tyrosine residues. Insulin (10(-7) M) stimulated the reaction by 4-10-fold by increasing Vmax with little change in Km. alpha-Tubulin was preferred as a substrate for the kinase compared to beta-tubulin. MAP2 was found to be the best substrate among the cytoskeletal proteins tested; in the presence of insulin, the Vmax for MAP2 was 6.3 nmol/min/mg, its Km was 5.1 microM, and 1.7 mol of phosphate were incorporated per mol of MAP2. Under the same conditions used for this phosphorylation of tubulin and MAPs, actin and tropomyosin were very poorly phosphorylated. These data, coupled with previous evidence for potential functional relationships between insulin action and microtubules, raise the possibility that microtubule proteins may be cellular targets for the insulin receptor kinase.

Humans↗
PubMed1985

Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase.

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.

Actins↗
PubMed1985

Characteristics of insulin receptors and insulin action in human myelogenous leukemia cell line K-562.

Specific binding sites for insulin have been identified and characterized for the human erythroleukemia cell line K-562. The binding of [125I]-insulin to the cells increased as a function of time, reaching a maximum at 20 min when incubation was performed at 37 degrees C. The binding of [125I]-insulin was dose-dependently inhibited by insulin or proinsulin. Scatchard plot of the binding data was curvilinear, and the number of insulin receptors was approximately 39,000. Insulin at concentrations of 0.05-10.0 ng/ml stimulated CO2 production and DNA and protein synthesis in K-562 cells in a dose-dependent manner, indicating that the insulin binding sites are functionally important in mediating these biochemical events induced by insulin. Maximal insulin responses were elicited at concentrations of less than 5 ng/ml, when (at most) 10% of the insulin receptors were occupied. After binding to the cells, [125I]-insulin was degraded in a time- and temperature-dependent manner. As reported for other types of cells, unlabeled insulin also downregulated insulin receptors in K-562 cells. When the cells were incubated with 1 X 10(-7) M unlabeled insulin for 24 h, the number of insulin receptors decreased by 50% without a change of affinity. K-562 cells may be useful in studying the role of insulin receptors in cell functions induced by insulin.

Carbon Dioxide↗
PubMed1985

[Phosphorylation of the receptor insulin and insulin-like growth factors].

We have found that the beta-subunit of the insulin receptor is a phosphoprotein and that the degree of phosphorylation is increased by the binding of insulin to its receptor. Furthermore we have found that the beta-subunit of the insulin receptor itself is a tyrosine-specific protein kinase. Tyrosine-specific protein kinases have been reported to be associated with the transforming gene product of RNA tumor viruses, the EGF receptor, the IGF-I receptor and a protein believed to be the receptor for PDGF. These findings suggest that determination of the endogenous substrated for these tyrosine-specific protein kinases may yield a sequence of regulatory proteins for cell growth and transformation. From this point of view, our recent findings that the purified insulin receptor-kinase can phosphorylate purified microtubule protein (tubulin, MAP2, tau) are interesting.

Animals↗
PubMed1984

Decreased autophosphorylation of the insulin receptor-kinase in streptozotocin-diabetic rats.

It has been documented that streptozotocin-induced diabetes in rats is associated with diminished effects of insulin despite increased insulin binding to its receptor. This paradox led us to examine whether any alterations of insulin receptor-kinase activities occur in this type of insulin resistance. Insulin binding capacity/mg of protein of solubilized, wheat germ agglutinin-purified preparations from livers was increased by 1.8-fold in the streptozotocin (65 mg/kg) diabetic rats. This increase was associated with a parallel increase in receptor protein as measured by an immunoblotting method using anti-insulin receptor antibody. Moreover, no apparent change was observed in the stoichiometry of alpha and beta subunits of the insulin receptor between diabetic and control rats. Insulin-stimulated (10(-7) M) phosphorylation of the beta subunit of the insulin receptor was decreased by 40% in diabetic rats when equal quantities of insulin binding capacity were compared. Phosphorylation of an exogenously added synthetic peptide (similar in sequence to the tyrosine phosphorylation site in pp60src) by the insulin receptor-kinase was also decreased by 25% in diabetic rats. These abnormalities were partially restored by in vivo insulin treatment. These data suggest that diminished insulin receptor autophosphorylation and kinase activity could provide a possible mechanism for the "post-binding insulin resistance" in diabetic rats.

Animals↗
PubMed1984

Insulin receptor regulation and desensitization in rat hepatoma cells. The loss of the oligomeric forms of the receptor correlates with the change in receptor affinity.

We have previously reported that prolonged incubations of Fao cells, a cell line derived from the well-differentiated Reuber H35 rat hepatoma, with 10(-6) M insulin, induced a decrease in receptor number (down-regulation), an increase in receptor affinity for insulin, and a loss of insulin's biological effect (desensitization). In the present study, we have investigated the relationship between these changes in insulin binding and action and changes in the structure of the insulin receptor. Intact cells were surface labeled with Na125I and lactoperoxidase, and the 125I-labeled insulin receptor was immunoprecipitated using specific antibodies and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of gels done under reducing conditions demonstrated the alpha (Mr = 135,000) and the beta (Mr = 95,000) subunits of the receptor. In nonreduced gels, free insulin receptor subunits were observed as well as four higher molecular weight bands with Mr = 210,000, 270,000, 350,000, and 520,000. Two-dimensional gel electrophoresis revealed that these bands correspond to alpha-beta heterodimer, alpha 2 homodimer, and two alpha-beta oligomers of high molecular weights, respectively. Cross-linking of 125I-insulin to intact cells with disuccinimidyl suberate revealed bands of Mr = 125,000, 210,000, 250,000 and 320,000, indicating that most of the forms of the receptor could bind insulin. After incubation with 10(-6) M insulin for 24 h, Fao cells revealed a marked decrease of the four oligomeric forms of the receptor, with little change in the level of the free alpha and beta subunits. A similar decrease of the oligomeric forms of the insulin receptor and an increase in the free subunits was observed when normal Fao cells are treated with 7 mM dithiothreitol. In dithiothreitol-treated cells, 125I-insulin binding was increased and this increase was accounted for by a change in affinity. In contrast to Fao cells, down-regulation of the insulin receptor in IM-9 lymphocytes occurs without a change in receptor affinity. In these cells, surface labeling revealed a decrease in total receptors after down-regulation, but not change in the proportion of the oligomeric forms to the free subunits of the receptor. These data suggest the following in Fao hepatoma cells. In the native state, the insulin receptor consists of free alpha and beta subunits and several kinds of disulfide-linked oligomers of these subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Site-specific antibodies to the erbB oncogene product immunoprecipitate epidermal growth factor receptor.

Antiserum to a defined region (residues 373-383) of the erbB oncogene product immunoprecipitated a 170,000 dalton protein that was phosphorylated in an EGF-sensitive fashion as well as the 125I-EGF-receptor complex from A431 human epidermoid carcinoma cells. Preincubation of the antiserum with an excess of the synthetic peptide corresponding to the defined region blocked the immunoprecipitation of this protein. A partial proteolytic peptide map of this immunoprecipitated 170,000 dalton protein was identical to that of the authentic EGF receptor. These results suggest immunological similarity between the erbB gene product and the EGF receptor.

Alpharetrovirus↗
PubMed1984

Phosphorylation and dephosphorylation of the insulin receptor: evidence against an intrinsic phosphatase activity.

We have studied the reversibility of insulin receptor phosphorylation to establish the relation between this autophosphorylation reaction and the initiation of insulin action and between dephosphorylation and the termination of insulin effects in cells. In cultured Fao hepatoma cells labeled with 32PO4(3-), insulin increased 5-fold the phosphorylation of the beta-subunit of the insulin receptor at serine, threonine, and tyrosine residues. Addition of anti-insulin antiserum to cells incubated with insulin caused dissociation of insulin from the receptor and concurrent dephosphorylation of the beta-subunit. 32PO4(3-) associated with the insulin-stimulated receptor could be decreased by the addition of sodium phosphate to the medium but with a slower time course. Insulin stimulated phosphorylation of insulin receptor purified partially on immobilized wheat germ agglutinin. This reaction utilized [gamma-32P] ATP and occurred exclusively on tyrosine residues. Addition of unlabeled ATP caused a decrease in the amount of PO4(3-) associated with the receptor. Insulin-stimulated phosphorylation was also observed if the receptors were further purified by immunoprecipitation with anti-insulin receptor antibody prior to the phosphorylation reaction; however, addition of unlabeled ATP to this system did not chase the labeled 32PO4(3-) from the beta-subunit. These data are consistent with the notion that phosphorylation and dephosphorylation of the insulin receptor parallel the onset and termination of insulin action. Phosphatase activity involved in the dephosphorylation of the insulin receptor appears to be a glycoprotein because it was retained after partial purification of the receptor on wheat germ agglutinin-agarose; however, this phosphatase activity is distinct from the insulin receptor because it was not retained after immunoprecipitation of the receptor with anti-insulin receptor antibodies.

Animals↗
PubMed1984

Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells.

Autophosphorylation of the insulin receptor was studied using a glycoprotein fraction solubilized and purified partially from the rat hepatoma cell line, Fao. Incubation of this receptor preparation with [gamma-32P] ATP, Mn2+, and insulin yielded a single insulin-stimulated phosphoprotein of Mr = 95,000 which corresponds to the beta-subunit of the insulin receptor. At 22 degrees C, incorporation of 32P was half-maximal at 30 s and about 90% complete after 2 min. At steady state, about 200 pmol of 32P were incorporated per mg of protein; this value corresponded to about 2 molecules of phosphate per insulin binding site estimated from Scatchard plots. Insulin increased the Vmax for autophosphorylation of the insulin receptor kinase nearly 20-fold with no effect on the Km for ATP. Mn2+ stimulated autophosphorylation by decreasing the Km of the kinase for ATP, whereas Mg2+ had no effect. Dilution of the insulin receptor over a 10-fold concentration range did not decrease the rate of autophosphorylation suggesting that it may occur by an intramolecular mechanism. When the phosphorylated beta-subunit of the insulin receptor was digested with trypsin, at least 5 phosphopeptides could be separated by high performance liquid chromatography on a mu Bondapak C18 reverse-phase column. Insulin stimulated the phosphorylation of all sites. These phosphate acceptor sites varied in their rate and degree of phosphorylation. Phosphopeptides pp4 and pp5 were phosphorylated very rapidly and reached steady state within 20 s, whereas phosphorylation of pp1 and pp2 required several minutes to reach steady state.

Adenosine Triphosphate↗
PubMed1984

Eradication of microscopic metastases with intratumoral injection of bacillus Calmette-Guerin.

The studies reported here were designed to examine the effects of intratumoral preoperative administration of Bacillus Calmette-Guerin (BCG) on the cure rates of C3H mice transplanted with MH134 tumor cells and on the metastatic rates in the regional lymph nodes. Furthermore, the morphological findings occurring in the regional lymph nodes were monitored during tumor growth using H-E stain and non-specific esterase staining. The cure rate of the Group treated with BCG intratumoral injection and surgery was significantly higher than that of the Group treated with surgery alone, and in the BCG + surgery group metastatic rates of regional lymph nodes decreased consistently after operation. Moreover, in this group, extensive sinus histiocytosis and marked swelling of the regional nodes were frequently observed. Quantitative studies of the cell kinds using the esterase staining indicated that intratumoral injection of BCG has an effect on the influx of lymphoid cells into the regional nodes, but does not aid specific cell lineage to flow into the regional nodes. In cytostatic assays, it was shown that the regional lymph node cells and spleen cells in the BCG + surgery group always have a greater per cent of inhibition than those in the surgery alone group.

Animals↗
PubMed1984

Immunosuppressive activity of sera from gastric cancer patients.

Sera from 60 gastric cancer patients and 20 patients with benign gastric diseases and 8 healthy controls were tested for inhibitory effects on the humoral response to sheep erythrocytes (SRBC) by the plaque forming cell assay (PFC R.I.) using mouse spleen cells and on the phytohemagglutinin (PHA)-induced blastogenesis of normal mouse spleen cells (PHA S.R.). Gastric cancer patient sera showed a significantly lower PFC R.I. than did sera from benign gastric disease patients and from the healthy controls. However, there was no appreciable interstage difference in the degree of depression. The PHA-induced blastogenesis of normal spleen cells was also decreased in the presence of sera from cancer patients, as compared to that in the presence of sera from benign disease patients and from the healthy controls. The depression progressed with advancing stage of cancer. The PHA S.R. showed significant negative correlations with serum levels of IAP, IS, alpha 1-acid glycoprotein and alpha 1-antitrypsin, but there were no such correlations between PFC R.I. and these glycoproteins in serum. There was also no correlation between the values of the PHA S.R. and the PFC R.I. These results suggest that these two assays may depict immunosuppressive activities operating through entirely different mechanisms.

Animals↗
PubMed1984

Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line.

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol-wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin-resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.

Binding, Competitive↗