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Biomedical subjects

M Seno

Publications and source records attributed to M Seno.

At least 37 records · Page 2Linked to original sources

Inhibition of cell growth by a fused protein of human ribonuclease 1 and human basic fibroblast growth factor.

Pancreatic-type RNases are considered to have cytotoxic potential due to their ability to degrade RNA molecules when they enter the cytosol. However, most of these RNases show little cytotoxicity because cells have no active uptake mechanism for these RNases and because the ubiquitous cytoplasmic RNase inhibitor is considered to play a protective role against the endocytotic leak of RNases from the outside of cells. To study the cytotoxic potential of RNase toward malignant cells targeting growth factor receptors, the C-terminus of human RNase 1 was fused to the N-terminus of human basic fibroblast growth factor (bFGF). This RNase-FGF fused protein effectively inhibited the growth of mouse melanoma cell line B16/BL6 with high levels of cell surface FGF receptor. This effect appeared to result from prolongation of the overall cell cycle rather than the killing of cells or specific arrest in a particular phase of the cell cycle. Thus, human RNase 1 fused to a ligand of cell surface molecules, such as the FGF receptor, is shown to be an effective candidate for a selective cell targeting agent with low toxic effects on normal cell types.

Amino Acid Sequence↗

Immunohistochemical localization of betacellulin, a new member of the EGF family, in normal human pancreas and islet tumor cells.

Betacellulin (BTC) purified from mouse beta cell tumor (betaTC-3) is a new member of the epidermal growth factor (EGF) family which can bind receptor tyrosine kinase, EGF receptor (erbB1) and erbB4. It has been demonstrated that proBTC mRNA was abundantly expressed in human pancreas tissue, and that BTC converted amylase-secreting rat acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC might be important for the growth and/or differentiation of islet cells. However, the cell type producing BTC in the pancreas has not been clarified. In this study, we examined the localization of BTC in human pancreas and islet cell tumors. Immunohistochemistry using specific antibodies to human BTC revealed that this protein was produced in alpha cells and duct cells, and probably in beta cells in normal adult pancreas. Furthermore, strong immunoreactivity to BTC was detected in primitive duct cells of the fetal pancreas, and both insulinoma and glucagonoma cells also showed positive immunoreactivity to BTC. EGF receptor (erbB1) and erbB4 were expressed mainly in islet and duct cells, and duct cells, respectively. These results demonstrate the localization of BTC and its receptors, and suggest that BTC may be one of the factors that have physiologically important roles such as growth and differentiation of islet cells in the human pancreas.

Adenoma, Islet Cell↗

Genes expressed during the differentiation of pancreatic AR42J cells into insulin-secreting cells.

Pancreatic AR42J cells have the feature of pluripotency of the common precursor cells of the pancreas. Dexamethasone (Dx) converts them to exocrine cells, whereas activin A (Act) converts them into endocrine cells expressing pancreatic polypeptide. A combination of Act and betacellulin (BTC) converts them further into insulin-secreting cells. The present study identifies some of the genes involved in the process of differentiation that is induced by these factors, using the mRNA differential display and screening of the cDNA expression array. The expression levels of 7 genes were increased by Act alone, and a combination of Act and BTC increased the expression of 25 more genes. Of these, 16 represented known genes or homologues of genes characterized previously. Nine of the identified genes were unrelated to any other sequences in the database. An inhibitor of the mitogen-activated protein kinase pathway, PD098059, which blocks the differentiation into insulin-secreting cells, inhibited the expression of 18 of the 25 genes, suggesting that the proteins encoded by these genes are associated with the differentiation into insulin-producing cells. These include known genes encoding extracellular signaling molecules, such as parathyroid hormone-related peptide, cytoskeletal proteins, and intracellular signaling molecules. Identification and characterization of these differentially expressed genes should help to clarify the molecular mechanism of differentiation of pancreatic cells and the gene products that enable the beta-cells to produce insulin.

Activins↗

BALB/c 3T3 cells co-expressing FGF-2 and soluble FGF receptor acquire tumorigenicity.

The physiological significance of the soluble fibroblast growth factor (FGF) receptors is not clear yet although they are present in blood, vitreous fluid and in the extracellular matrix of vascular endothelial cells. A hypothesis that they might help FGF-2 release from cells is very interesting because FGF-2 does not have clear secretion signal and the mechanism of the secretion of FGF-2 is still unclear. Single overexpression of FGF-2 is related neither to the secretion potential of the molecule nor to the tumorigenicity of the cells. In this report, BALB/c 3T3 cells transformed with the full length of human FGF-2 cDNA are further transformed with the cDNA coding the extracellular domain of human FGF receptor 1. The obtained transformants co-expressing FGF-2 and soluble FGF receptor are highly tumorigenic in nude mice, while the parental cells do not show any tumorigenicity. In the conditioned medium of the double-transformants, FGF-2 is immunologically detected. These results suggest that naturally produced soluble form of FGF receptor supports the release of FGF-2 from the cells and that over-expression of these two molecules leads to induce the malignant tumours in vivo.

3T3 Cells↗

Studies on the betacellulin receptor in pancreatic AR42J cells.

Betacellulin is a member of the epidermal growth factor family and converts pancreatic AR42J cells into insulin-producing cells. This study was conducted to characterise the receptor for betacellulin in AR42J cells. AR42J cells expressed two classes of binding sites for radioactive iodine labelled betacellulin, with Kd values of 4.6 x 10(-11) mol/l and 3.0 x 10(-10) mol/l. The binding of [125I]betacellulin was inhibited by unlabelled betacellulin in a dose-dependent manner, but epidermal growth factor was 50 fold less effective than betacellulin. Affinity cross-linking showed a [125I]betacellulin-binding protein with a molecular weight of approximately 180 KDa. When this protein was immunoprecipitated with antibody against epidermal growth factor receptors ErbB-1, ErbB-2, ErbB-3 or ErbB-4, it was immunoprecipitated only by the anti-ErbB-1 antibody. When the [125I]betacellulin-labelled proteins were immunoprecipitated with a combination of the four ErbB antibodies, and the unprecipitated proteins were then immunoprecipitated with anti-phosphotyrosine antibody, a 190 KDa protein was observed. Betacellulin induced the tyrosine phosphorylation of ErbB-1, ErbB-2 and ErbB-4. Finally, while 100 pmol/1 betacellulin converted all of the AR42J into insulin-producing cells in the presence of activin A, 10 nmol/l epidermal growth factor induced differentiation in only about 30 % of the cells. Higher concentrations of epidermal growth factor were less effective. Neu differentiation factor in the presence or absence of epidermal growth factor was ineffective. These results indicate that betacellulin binds to ErbB-1 and possibly another protein with a molecular weight of 190 KDa. The latter betacellulin-binding protein may be involved in the differentiation-inducing activity of betacellulin.

Animals↗

Human pancreatic RNase1-human epidermal growth factor fusion: an entirely human 'immunotoxin analog' with cytotoxic properties against squamous cell carcinomas.

The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with a gene encoding human epidermal growth factor (hEGF). The hybrid human protein was isolated from Escherichia coli inclusion bodies, refolded and purified to homogeneity. The fusion protein competed with 125I-hEGF for binding to hEGF receptors (EGFR) and had ribonucleolytic activities approaching those of hpRNase1. Several conformations having different enzymatic activities could be detected after reversed-phase high-performance liquid chromatographic analysis, the less hydrophobic molecules being the most active. The hybrid protein was specifically cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line, with an IC50 of approximately 10(-7) M. In contrast, recombinant hpRNase1 had an IC50 higher than 10(-4) M. A mixture of free hEGF and free hpRNasel was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected in EGFR-deficient control cells. Taken together, these data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast cancers overexpressing EGFR, which correlate with a poor prognosis and cannot be cured by surgery alone. Engineering hybrid molecules with endogenous human proteins for targeted therapy may alleviate the dose-limiting immunogenicity and toxicity of conventional immunotoxins.

Binding, Competitive↗

Electron microscopic study of intercalated duct cells in the chicken pancreatic islet and effects of tolbutamide administration.

Intercalated duct cells are present in the alpha and beta islets of chicken. The intercalated duct cells adhere to each other via intercellular junctional complexes at the apical side, projecting many microvilli and a few cilia into the lumen. They also extend slender cytoplasmic processes between the islet endocrine cells. These intercalated cells appear to have a stellate form, and to wrap their cytoplasm around endocrine cells. Administration of tolbutamide led to increased electron density in the cytoplasm of intercalated duct cells. Lysosomes are present in these cells in various numbers and sizes and tend to increase with time after administration of tolbutamide. These observations suggested that the intercalated ducts not only pass through the islet, but also play a role in supplying islet cells.

Animals↗

Purification and characterization of a recombinant human cripto-1 protein.

Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.

Animals↗

Molecular Targeting for Epidermal Growth Factor Receptor Expressed on Breast Cancer Cells by Human Fusion Protein.

Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The cytotoxicity of this conjugate was assayed using MTT assay. The EGF-RNase conjugate showed dose-dependent cytotoxicity against breast and squamous cell carcinomas overexpressing the EGF receptor (EGFR). The cytotoxicity of the conjugate correlated positively with the level of EGFR expression by each cell line. These results suggest that the EGF-RNase conjugate is a more effective anticancer agent with less immunogenicity and toxicity than conventional chimeric breast cancer toxins.

Journal Article↗

Cripto enhances the tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase (MAPK) in mammary epithelial cells.

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor alpha, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin beta1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42(erk-2) mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Animals↗

Tissue-specific expression of pancreatic-type RNases and RNase inhibitor in humans.

The tissue-specific expression of five human pancreatic-type RNases and RNase inhibitor was analyzed by Northern hybridization against poly(A)+ RNA prepared from 16 normal tissues. The widespread expression of RNase 1 was observed in almost all of the tissues. RNase 4 and angiogenin showed a similar distribution of expression abundantly present in the liver. This suggested the identity of the cell types producing these two molecules. However, no relativity appeared to be present between the vascularization of the tissues and the angiogenin expression. A narrow range of expression of the eosinophil-derived neurotoxin gene was observed. This localization seems related to the phagocytic cells in the tissues. The undetectable level of the eosinophil cationic protein mRNA in normal tissues suggests that the differentiation of eosinophils, triggered by inflammation and/or atopy, is required. The expression of RNase inhibitor was found to be ubiquitous. The regulatory function of inhibitor against RNases in the cell should be considered in studying the physiological significance of the pancreatic-type RNase family.

DNA, Complementary↗

Protection of mice from respiratory Sendai virus infections by recombinant vaccinia viruses.

Mechanisms of protection of mice from Sendai virus, which is exclusively pneumotropic and causes a typical respiratory disease, by immunization with recombinant vaccinia viruses (RVVs) were investigated. Although the RVV carrying a hemagglutinin-neuraminidase gene of Sendai virus (Vac-HN) propagated in the noses and lungs of mice by either intranasal (i.n.) or intraperitoneal (i.p.) inoculation, no vaccinia virus antigens were detected in the mucosal layer of upper and lower airways of the i.p.-inoculated mice. The mice immunized i.n. with Vac-HN or Vac-F (the RVV carrying a fusion protein gene of Sendai virus) demonstrated the strong resistance to Sendai virus challenge both in the lung and in the nose, whereas the i.p.-immunized mice showed almost no resistance in the nose but showed a partial resistance in the lung. Titration of Sendai virus-specific antibodies in the nasal wash (NW), bronchoalveolar lavage (BAL), and serum collected from the Vac-F-immunized mice showed that the NW from the i.n.-immunized mice contained immunoglobulin A (IgA) antibodies but no IgG and the BAL from the mice contained both IgA and IgG antibodies. On the other hand, neither IgA nor IgG antibodies were detected in the NW from the i.p.-immunized mice and only IgG antibodies were detected in the BAL, although both i.n.- and i.p.-immunized mice exhibited similar levels of serum IgG, IgA, and neutralizing antibodies. The resistance to Sendai virus in the noses of i.n.-immunized mice could be abrogated by the intranasal instillation of anti-mouse IgA but not of anti-IgG antiserum, while the resistance in the lung was not significantly abrogated by such treatments. These results demonstrate that IgA is a major mediator for the immunity against Sendai virus induced by the RVVs and IgG is a supplementary one, especially in the lung, and that the RVV should be intranasally inoculated to induce an efficient mucosal immunity even if it has a pantropic nature.

Administration, Intranasal↗

Cripto-1 inhibits beta-casein expression in mammary epithelial cells through a p21ras-and phosphatidylinositol 3'-kinase-dependent pathway.

Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that does not directly activate any of the known erbB type 1 tyrosine kinase receptors. Also, CR-1 stimulates the growth of HC-11 mouse mammary epithelial cells. We found that prior treatment of HC-11 cells with exogenous CR-1 induced a competency response to the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) with respect to the induction of the milk protein beta-casein. In contrast, simultaneous treatment of mouse HC-11 cells with CR-1 in the presence of DIP inhibited beta-casein expression. The inhibitory effects of CR-1 on beta-casein expression in response to DIP were not unique to this mouse mammary epithelial cell line, because beta-casein and whey acidic protein expression in primary mouse mammary explant cultures established from midpregnant mice were also differentially inhibited by several epidermal growth factor-related peptides including CR-1. The mitogenic and differentiation effects of CR-1 are mediated by the binding of CR-1 to a cell surface receptor that is known to activate the ras/raf/mitogen-activated protein kinase (MAPK)/MAPK kinase pathway. The inhibitory response of CR-1 in HC-11 cells on beta-casein expression after treatment with DIP can be attenuated by B581, a peptidomimetic farnesyltransferase inhibitor that blocks p21ras farnesylation and activation, and by the phosphatidylinositol 3'-kinase (PI3k) inhibitor LY 294002 but not by PD 98059, a MAPK kinase inhibitor that blocks MAPK activation. These data suggest that the ability of CR-1 to block lactogenic hormone-induced expression of beta-casein is mediated through a p21ras-dependent, PI3k-mediated pathway. This is further substantiated by the observation that CR-1 is able to stimulate the tyrosine phosphorylation of the p85 PI3k regulatory subunit and to increase the activity of PI3k in HC-11 cells.

Animals↗

Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells.

Rat pancreatic AR42J cells possess exocrine and neuroendocrine properties. Activin A induces morphological changes and converts them into neuron-like cells. In activin-treated cells, mRNA for pancreatic polypeptide (PP) but not that for either insulin or glucagon was detected by reverse transcription-PCR. About 25% of the cells were stained by anti-PP antibody. When AR42J cells were incubated with betacellulin, a small portion of the cells were stained positively with antiinsulin and anti-PP antibodies. The effect of betacellulin was dose dependent, being maximal at 2 nM. Approximately 4% of the cells became insulin positive at this concentration, and mRNAs for insulin and PP were detected. When AR42J cells were incubated with a combination of betacellulin and activin A, approximately 10% of the cells became insulin positive. Morphologically, the insulin-positive cells were composed of two types of cells: neuron-like and round-shaped cells. Immunoreactive PP was found in the latter type of cells. The mRNAs for insulin, PP, glucose transporter 2, and glucokinase, but not glucagon, were detected. Depolarizing concentration of potassium, tolbutamide, carbachol, and glucagon-like peptide-1 stimulated the release of immunoreactive insulin. These results indicate that betacellulin and activin A convert amylase-secreting AR42J cells into cells secreting insulin. AR42J cells provide a model system to study the formation of pancreatic endocrine cells.

Activins↗

Elevated erythrocyte sodium-lithium countertransport activity correlates with increased intracellular sodium and free calcium-ion concentration in type 2 diabetes.

To explore the mechanism by which elevated sodium-lithium countertransport activity (SLC) associates with both predisposition to hypertension and diabetic nephropathy, we investigated the interrelationships among SLC, intracellular sodium concentration ([Na]i), intracellular free calcium-ion concentration ([Ca2+]i), diabetic nephropathy, hypertension, family history of hypertension, and other factors in 48 patients with Type 2 diabetes and 24 healthy controls. There was a significant correlation between SLC and [Na]i (r = 0.36, p < 0.05), [Na]i and [Ca2+]i (r = 0.47, p < 0.01), SLC and [Ca2+]i (r = 0.46, p < 0.01), and between HbA1c and [Ca2+]i (r = 0.38, p < 0.01). Both [Na]i and [Ca2+]i were significantly higher in the patients with elevated SLC than in those with normal SLC (p < 0.01 and p < 0.05, respectively). In stepwise multivariate regresssion analysis, [Na]i, HbA1c, and SLC appeared as independent determinants of [Ca2+]i. These data suggest a significant correlation of elevated SLC with increased [Na]i and [Ca2+]i. This may be a possible mechanism underlying the close association of elevated SLC with both predisposition to hypertension and diabetic nephropathy.

Adult↗

Epidermal growth factor receptor-dependent cytotoxicity for human squamous carcinoma cell lines of a conjugate composed of human EGF and RNase 1.

Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The EGF-RNase conjugate showed dose-dependent cytotoxicity for EGF receptor-overexpressing A431 and TE-8 human squamous carcinoma cells with an IC50 of 2 x 10(-7)M and 10(-6)M, respectively, whereas the IC50 of RNase alone was almost 10(-4)M. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone. The conjugate showed no detectable cytotoxicity against EGF receptor-deficient small cell lung cancer cells (H69). Addition of excess EGF in the medium protected A431 cells from the EGF-RNase conjugate cytotoxicity. The cytotoxicity of the EGF-RNase conjugate was positively correlated with the EGF receptor numbers of each cell line. The chimeric toxin composed of only human proteins might be a more useful anti-cancer agent with less immunogenicity than the conventional chimeric toxins.

Carcinoma, Squamous Cell↗

Human betacellulin, a member of the EGF family dominantly expressed in pancreas and small intestine, is fully active in a monomeric form.

Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine. This finding suggests that BTC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family. To clarify this function, the released form of human BTC has been expressed in E.coli, purified, and characterized. The recombinant human BTC was produced as an inclusion body. This material was dissolved in guanidine-HCl under reducing conditions, refolded, and purified through sequential liquid chromatography. Purified BTC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide. However, chemical analysis failed to show a covalently linked dimer. The molecular mass of BTC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer. Recombinant BTC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF On the other hand, BTC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor.

3T3 Cells↗

Recombinant human pancreatic ribonuclease produced in E. coli: importance of the amino-terminal sequence.

Human pancreatic ribonuclease 1 (hRNase 1) in the mature form has been produced in E. coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase 1 to the characteristics of the protein.

Amino Acid Sequence↗