Hitomi Murakami

Transforming growth factor (TGF) - α in human milk

Abstract Transforming growth factor (TGF) - α and epidermal growth factor (EGF) were measured in human milk by means of homologous radioimmunoassay. As previously reported, EGF concentration in the colostrum was approximately 200 ng/ml and decreased to 50 ng/ml by day 7 postpartum. The value of immunoreactive (IR) -TGF- α was 2.2–7.2 ng/ml, much lower than that of EGF. In contrast to EGF, the concentration of IR-TGF-α was fairly stable during the 7 postpartum days. There was no relationship between the concentrations of IR-TGF-α and IR-EGF, suggesting that the regulatory mechanism in the release of the two growth factors is different. On gel-chromatography using a Sephadex G-50 column, IR-EGF appeared in the fraction corresponding to that of authentic human EGF, while 70%–80% of the IR-TGF-α was eluted as a species with a molecular weight greater than that of authentic human TGF-α. Although the physiological role of TGF-α in milk is not known, it is possible that it is involved in the development of the mammary gland and/or the growth of newborn infants.

Potentiation of Mitogenic Activity of Platelet-Derived Growth Factor by Physiological Concentrations of Insulin via the MAP Kinase Cascade in Rat A10 Vascular Smooth Muscle Cells

Hyperinsulinemia has been shown to be associated with diabetic angiopathy. Migration and proliferation of vascular smooth muscle cells (VSMC) are the processes required for the development of atherosclerosis. In this study, we attempted to determine whether insulin affects mitogenic signaling induced by plateletderived growth factor (PDGF) in a rat VSMC cell line (A10 cells). PDGF stimulated DNA synthesis which was totally dependent on Ras, because transfection of dominant negative Ras resulted in complete loss of PDGF-stimulated DNA synthesis. Initiation of DNA synthesis was preceded by activation of Raf-1, MEK and MAP kinases (Erk 1 and Erk2). Treatment of the cells with PD98059, an inhibitor of MAPK kinase (MEK) attenuated but did not abolish PDGF-stimulated DNA synthesis, suggesting that MAPK is required but not essential for DNA synthesis. PDGF also stimulated phosphorylation of protein kinase B (Akt/PKB) and p70 S6Kinase (p70S6K) in a wortmannin-sensitive manner. Rapamycin, an inhibitor of p70S6K, markedly suppressed DNA synthesis. Low concentrations of insulin (1-10 nmol/l) alone showed little mitogenic activity and no significant effect on MAPK activity. However, the presence of insulin enhanced both DNA synthesis and MAPK activation by PDGF. The enhancing effect of insulin was not seen in cells treated with PD98059. Insulin was without effect on PDGF-stimulated activations of protein kinase B (Akt/PKB) and p70S6K. We conclude that insulin, at pathophysiologically relevant concentrations, potentiates the PDGFstimulated DNA synthesis, at least in part, by potentiating activation of the MAPK cascade. These results are consistent with the notion that hyperinsulinemia is a risk factor for the development of atherosclerosis.

Demonstration of Heterogeneity of Autoantibodies to Insulin Receptors in Type B Insulin Resistance by Isoelectric Focusing

With isoelectric focusing, we examined heterogeneity of autoantibodies to insulin receptors in serums of two patients with insulin-resistant diabetes and one patient with hypoglycemia. Immunoglobulins were prepared by ammonium sulfate precipitation and ion-exchange chromatography with DEAE-Sepharose and subjected to isoelectric focusing for separation into 30 fractions. The fractions were tested for their ability to inhibit 125I-labeled insulin binding to human placental membranes, immunoprecipitate solubilized insulin receptor cross-linked with 125I-insulin, and mimic or inhibit the action of insulin in rat adipocytes. The results varied among the three patients. In the first patient, inhibition of 125I-insulin-binding activity (IBA) and insulin-receptor-precipitating activity (IPA) were distributed almost identically, but the distribution of insulinlike bioactivity (ILBA) was somewhat different. In the second patient, some fractions exhibited potent IBA without IPA, and these fractions inhibited the action of insulin in rat adipocytes. In the third patient, all of the isoelectric fractions showed IBA without IPA and were insulin antagonists. These observations indicate that some patients have antibodies with pure insulin-antagonist properties and provide further evidence that autoantibodies to insulin receptors are polyclonal and recognize different antigenic sites on insulin-receptor molecules. The findings also suggest that the ability of antibodies to elicit ILBA is linked to the ability to immunoprecipitate 125I-insulin-cross-linked and solubilized receptors, whereas antibodies that only inhibit insulin binding behave as insulin antagonists.

Insulin-like growth factor-II (IGF-II)/Mannose-6-phosphate receptors are increased in primary human thyroid neoplasms

Insulin-like growth factor (IGF)-II receptors were demonstrated in normal and neoplastic tissues of human thyroid. Specific binding of [125I]IGF-II to thyroid membranes was dependent on the time and temperature of incubation, and a steady state was achieved after 22 h of incubation at 4 degrees C. The binding of [125I]IGF-II was dose-dependently displaced by unlabelled IGF-II with 50% inhibition at an IGF-II concentration of 6 ng/ml. IGF-I had a relative potency of 1% compared to IGF-II, and insulin showed no inhibition at concentrations as high as 2000 ng/ml. Scatchard analysis of the binding data revealed a single class of IGF-II receptor with high affinity. Affinity crosslinking and autoradiography demonstrated the type II IGF receptors. Specific binding of [125I]IGF-II to thyroid papillary cancer tissues (mean [S.D.]13.2[1.3]% per 200 micrograms protein, n = 8) was significantly (P less than 0.01) higher than that to the surrounding normal tissues (4.8 [0.5]%). The binding in follicular cancer and follicular adenoma was also significantly higher than that in the corresponding normal tissues. The higher IGF-II binding to neoplastic tissues was due to an increase in the number of binding sites without any change of affinity. These results suggest that the increased IGF-II receptors may be involved in growth or functions of thyroid neoplasms.

Differentiation of human promyelocytic leukemia cells is accompanied by an increase in insulin receptors

Abstract Changes in insulin receptors accompanying cell differentiation in human promyelocytic leukemia cells (HL-60) were studied. Cell differentiation was induced by 1α,25-dihydroxyvitamin D 3 , vitamin A, dimethyl sulfoxide, or phorbol esters. 1α,25-dihydroxy-vitamin D 3 increased the ability of HL-60 cells to bind insulin in a dose-dependent manner. The increase in insulin binding was due to an increase in the number of insulin receptors. Vitamin A, dimethyl sulfoxide and phorbol esters were also effective in increaseing insulin receptors. Thus, the differentiation of HL-60 cells was accompanied by an increase in insulin receptors.

Increased activity of insulin-like growth factor-binding protein in human thyroid papillary cancer tissue.

It has been shown that both insulin‐like growth factor‐I (IGF‐I) and IGF‐binding proteins (IGFBPs) are produced by thyroid cells in culture and that the cells respond to IGF‐I with increased DNA synthesis, suggesting an autocrine/paracrine role of IGF‐I in the regulation of thyroid cell growth. We investigated the tissue contents of immunoreactive IGF‐I (irIGF‐I) and IGFBPs in human papillary carcinoma and compared them with those of normal thyroid tissue. When irIGF‐I was measured after separation of the IGFBPs by gel‐filtration, its content in carcinoma tissue was not different from that in adjacent normal tissue (566±58 vs. 424±75 pg/mg protein, N = 10). Nor was there any difference in the abundance of IGF‐I mRNA expression determined by slot blot analysis. On the other hand, IGFBP activity measured in terms of 125I‐IGF‐I binding was significantly higher in cancer extracts. Western ligand blot analysis of IGFBPs revealed several species (24–42 kDa) of IGFBPs. The IGF‐I‐binding activity of 38–41 kDa species (corresponding to IGFBP‐3) was not different between extracts of cancer tissue and those of normal tissue, whereas that of 28–32 kDa species was significantly higher in cancer tissue extracts. Since IGFBPs have been reported to modulate cellular responses to IGF‐I, the present data suggest that higher IGFBP activity in cancer tissue is involved in regulating growth of thyroid papillary carcinoma cells.

Mechanism of inhibitory actions of minocycline and doxycycline on ascitic fluid production induced by mouse fibrosarcoma cells

Semisynthetic tetracyclines (TCNs) are used for the management of malignant pleural effusions as sclerosing agents. However, their precise mechanism of actions are uncertain. In the present study, the mechanism of inhibitory effects of minocycline (MINO) and doxycycline (DOXY), on the accumulation of ascitic fluid induced by mouse fibrosarcoma (Meth-A) cells were investigated using male mice. Meth-A cells inoculated intraperitoneally elicited 2.5-4 ml of bloody ascites 10 days after implantation. The production of ascitic fluid was suppressed in a dose-related manner by daily intraperitoneal injections of MINO or DOXY, whereas vehicle (normal saline with 0.01N HCl) did not exert a significant effect. The inhibitory activity of these two substances was quite similar; one mg/mouse of MINO or DOXY inhibited the accumulation of fluid by 87% and 84%, respectively. The survival rate of Meth-A-bearing mice treated with MINO or DOXY was higher than that of the controls. Macroscopic examination of the peritoneal cavity did not reveal any obvious effects, such as adhesions, in mice treated with either MINO or DOXY. In vitro studies showed that MINO and DOXY suppressed Meth-A cell growth with IC50s of 5 microM and 8 microM, respectively. Maximal suppression (95%) was achieved at MINO and DOXY concentrations of 25 microM. The above observations suggest that MINO and DOXY inhibit the accumulation of ascites by a direct effect on Meth-A cell growth. Therefore, it appears that TCNs injected into the pleural cavity to manage malignant effusions in man exert their activity, at least in part, by suppressing malignant cell growth.

Transforming growth factor - α activity in effusions: Comparison of radioimmunoassay and radioreceptorassay

Transforming growth factor-alpha (TGF-alpha) in pleural and peritoneal effusions was assayed by homologous radioimmunoassay (RIA) and radioreceptor assay (RRA) using human placental membrane. Effusions were obtained from 24 patients with and 17 patients without cancer. Most of the effusions were found to contain TGF-alpha by RIA and RRA, but immunoreactive epidermal growth factor (EGF) was not detected. Effusions were chromatographed on Bio-Gel P-60 with several peaks of TGF-alpha activity by both RIA and RRA. A discrepancy in the chromatographic pattern of TGF-alpha between RIA and RRa suggested the existence of EGF-like substances capable of binding to EGF receptors which lack immunoreactivity for EGF or TGF-alpha. The TGF-alpha concentration of the acetic acid-extracted malignant effusions assayed by RRA significantly exceeded the value obtained from benign effusions (17.0 +/- 8.7 vs. 9.2 +/- 6.3 ng/ml; mean +/- SD: p < 0.02). However, the concentrations obtained by RIA did not differ.