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S Kasayama

Publications and source records attributed to S Kasayama.

53 records · Page 3Linked to original sources
PubMed1993

Androgen-induced growth factor and its receptor: demonstration of the androgen-induced autocrine loop in mouse mammary carcinoma cells.

SC-3 cells derived from mouse mammary carcinoma (Shionogi carcinoma 115) exhibit remarkable growth enhancement and cell morphology change in response to androgen stimuli. These events are mediated through an androgen-induced growth factor (AIGF). Amino acid sequence deduced from cDNA reveals that AIGF has 215 amino acids with a signal peptide and scattered regions homologous to fibroblast growth factor (FGF) family proteins. The biological ability of AIGF to stimulate SC-3 cell growth is inhibited by heparin or suramin. More importantly, antisense oligodeoxynucleotide of AIGF can block androgen-induced growth of SC-3 cells. Upon synthesis under the control of androgen, AIGF is immediately secreted into the extracellular space without intracellular accumulation. At the early phase (18-24 h) of androgen stimulation, however, AIGF is mainly associated with the glycosaminoglycan on the cell surface or extracellular matrix. In addition, treatment of SC-3 cells with sulfation blocker (chlorate) or heparitinase results in the abolishment of their ability to respond to androgen or AIGF, indicating that heparan sulfate has important roles for condensing AIGF on or near the cell surface as well as potentiating the biological activity of AIGF. Then, AIGF can bind to the FGF receptor. Northern blot analysis and cDNA cloning indicate that SC-3 cells predominantly express the FGF receptor 1 with some altered amino acid sequences. Transfection of expression vectors of AIGF and this variant form of FGF receptor 1 into FGF receptor-negative myoblast cells (L 6 cells) confirms that a variant form of FGF receptor 1 is a receptor of AIGF. These results clearly demonstrate that an autocrine mechanism is operating in androgen-induced growth of SC-3 cells.

Amino Acid Sequence↗
PubMed1993

A role for heparan sulfate in androgen-induced deoxyribonucleic acid synthesis of mouse mammary carcinoma (Shionogi carcinoma 115)-derived SC-3 cells.

In response to androgen stimuli, SC-3 cells cloned from mouse mammary carcinoma (Shionogi carcinoma 115) secrete heparin-binding growth factor, which is able to bind to fibroblast growth factor (FGF) receptor, ultimately resulting in SC-3 cell proliferation. A role for glycosaminoglycan in the process of this androgen-induced autocrine loop was examined. When quiescent SC-3 cells were stimulated with testosterone, enhanced DNA synthesis was demonstrated even at the early phase (12-24 h) of stimulation. During this early period, autocrine growth-promoting activity was found to be associated with cell surface and extracellular matrix, but not to be present in the conditioned medium. This bound form of growth-promoting activity was able to be extracted with 2 M NaCl (pH 7.5) and absorbed onto a heparin-Sepharose affinity column, from which it was eluted at a concentration of 1.1-1.3 M NaCl. Extracted growth factor, whose activity was partially blocked by anti-basic FGF antibody, up-regulated the expression of FGF receptor-1 mRNA. These characteristics were similar to those of a soluble form of SC-3-derived growth factor previously reported from our laboratory. Androgen-induced enhancement of DNA synthesis was inhibited by simultaneous treatment of SC-3 cells with 10 mIU/ml heparitinase or 25 mM sodium chlorate (an inhibitor of phosphoadenosine sulfate synthesis). However, chlorate treatment did not affect the synthesis and distribution of androgen-induced growth factor, suggesting that the response of chlorate-treated cells to this growth factor was impaired. These results indicate that heparan sulfate has important roles in concentrating androgen-induced heparin-binding growth factor on or very close to the cell surface and in potentiating its bioactivity.

Animals↗
PubMed1992

Characterization of the promoter region of the murine fibroblast growth factor receptor 1 gene.

To obtain some clue for the regulatory mechanism by which fibroblast growth factor (FGF) receptor 1 (FGFR 1) gene is expressed, we have cloned the promoter region of this gene from genomic library of mouse FGF-responsive cell lines. The genomic clone isolated here includes the FGFR 1 gene from position -868 to +697 relative to transcription initiation site. Sequence analysis reveals the presence of various consensus sequences for the binding sites of transcriptional factors such as SP 1, GCF, Oct-I, AP 1 and AP 2, but the absence of TATA and CAAT sequence motif. The transfection of this promoter-CAT constructs into NIH 3T3 cells demonstrates its promoter activity which is at least located between base -106 and +104.

3T3 Cells↗
PubMed1992

Identification and characterization of high molecular weight forms of basic fibroblast growth factor in human pituitary adenomas.

Basic fibroblast growth factor (bFGF) extracted from 23 human pituitary adenomas and 2 nontumorous anterior pituitary glands was examined by Western blot analysis to elucidate their molecular forms. Four bands that specifically interacted with anti-bFGF antibody were identified at the regions of 16K, 18K, 22K, and 24K mol wt. These immunodetectable bFGFs were bound to a heparin-Sepharose affinity column and eluted with 1-2 M NaCl. These partially purified tumor extracts could stimulate dose-dependently the growth of FGF-responsive cells. Some tumors contained bFGF with a mol wt of 24 K as a dominant form. In contrast, a major form of bFGF at the region of 18K was identified in the nontumorous anterior pituitary glands. We also addressed the possible presence of FGF receptor mRNA using Northern blot analysis. Many, but not all, human pituitary adenomas were found to contain detectable levels of FGF receptor mRNA with a size of 3.5 kilobases. The present results suggest that the role of bFGF in growth and hormone secretion in human pituitary adenomas should be examined using high mol wt forms of bFGF.

Adenoma↗
PubMed1991

Effects of thyroid hormone on androgen- or basic fibroblast growth factor-induced proliferation of Shionogi carcinoma 115 mouse mammary carcinoma cells in serum-free culture.

DNA synthesis of SC-3 cells cloned from mouse mammary carcinoma (Shionogi carcinoma 115) was remarkably enhanced by androgen as well as basic fibroblast growth factor (bFGF) at the early phase (days 1-3) of stimulation in serum-free culture condition. However, bFGF-induced DNA synthesis could not be observed at the late phase (days 4-6) of stimulation while androgen was able to continuously elicit DNA synthesis. When the effect of androgen on cell yield was examined, the cell number was increased while bFGF could not enhance cell growth. Androgen-induced heparin-binding growth factor partially purified from conditioned medium behaved like bFGF in terms of DNA synthesis and replication in SC-3 cells. SC-3 cells were found to contain the high-affinity binding site toward triiodothyronine. The dissociation constant and the maximum number of the binding sites were 7 x 10(-10) M and 1800/cell, respectively. Triiodothyronine significantly blunted the testosterone-induced DNA synthesis. On the other hand, bFGF-enhanced DNA synthesis was not substantially inhibited by triiodothyronine. These results suggest that androgen, but not bFGF, has unique action site(s) which might be important for SC-3 cell replication and might be antagonized by thyroid hormone.

Animals↗
PubMed1991

Expression cDNA cloning of fibroblast growth factor (FGF) receptor in mouse breast cancer cells: a variant form in FGF-responsive transformed cells.

Expression cDNA library of fibroblast growth factor (FGF)-responsive mouse breast cancer cells (SC-3) was prepared and screened using chick FGF receptor (FGF-R) cDNA as a probe. Two positive clones were isolated. Sequence analysis revealed that two clones have an identical full open reading frame. Compared with sequence data on mouse brain FGF-R, SC-3 cells were found to contain FGF-R with 12 amino acids insertion near N-terminal region. This insertion was mainly composed of hydrophobic amino acids. Additionally, the deletion of two amino acids in extracellular domain and the substitution of one amino acid in C-terminal region were identified in SC-3 cell FGF-R. Transfection of this clone into CHO cells resulted in a significant increase in basic FGF (bFGF) binding.

Amino Acid Sequence↗
PubMed1991

Up-regulation of fibroblast growth factor (FGF) receptor mRNA levels by basic FGF or testosterone in androgen-sensitive mouse mammary tumor cells.

Since we had previously shown that both basic fibroblast growth factor (bFGF) and testosterone stimulate the growth of mouse mammary carcinoma cells (SC-3) in serum-free culture, we tested the effect of bFGF or testosterone on FGF receptor mRNA levels. Northern blot analyses revealed that stimulation with bFGF resulted in a 5-fold increase in FGF receptor mRNA levels at 6-8 h followed by a decline to the unstimulated levels at 24 h. Simultaneous addition of cycloheximide blocked bFGF-induced accumulation of FGF receptor mRNA, although exposure of SC-3 cells to cycloheximide alone caused marginal increase in its basal level. Neither phorbol ester nor forskolin stimulated FGF receptor mRNA expression, but testosterone could raise FGF receptor mRNA levels. To obtain the maximum stimulation, however, testosterone required the longer stimulation period (12 h) than bFGF, suggesting that testosterone-induced FGF receptor mRNA accumulation is mediated through an induction of FGF-like growth factor.

Animals↗
PubMed1991

Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115.

An androgen-responsive cloned cell line (SC-3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)-like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC-3 cells in an autocrine mechanism. Since the androgen-induced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC-3 cells. Heparin was found to exhibit significant inhibition of testosterone-induced growth in a concentration-dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 micrograms/ml. DNA synthesis of SC-3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC-3 cells induced by acidic (a) or basic (b) FGF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen-induced growth factor in the conditioned medium from testosterone-treated cells. These results indicate that heparin inhibits autocrine loop in SC-3 cells induced by androgen. Thus, the autocrine growth factor possesses a different characteristic from aFGF and bFGF in that its bioactivities are negatively modulated by the glycosaminoglycan.

Animals↗
PubMed1991

Functional abnormality of glucocorticoid receptor in Shionogi carcinoma 115 cells as evidenced by gene transfer experiments.

The assay systems for steroid receptor functions in steroid-sensitive cells (SC-3 cells) were developed in which hormone-responsive element linked to a reporter gene [chloramphenicol acetyl transferase (CAT) gene] was transfected by the electroporation technique. Stimulation with androgen of SC-3 cells transfected with mouse mammary tumor virus promoter-CAT gene (MMTV-CAT) resulted in clear enhancement of CAT activity, whereas glucocorticoid required abnormally high concentrations to obtain significant stimulation. The simultaneous addition of glucocorticoid surprisingly inhibited androgen-induced CAT activity in SC-3 cells, whereas glucocorticoid and androgen acted together synergistically to activate CAT activity in T 47D cells. When SC-3 cells were cotransfected with the expression vector of human glucocorticoid receptor (GR) gene, inhibition with glucocorticoid of androgen-enhanced CAT activity was abolished. These results would suggest that SC-3 cells contain functionally abnormal GR.

Animals↗
PubMed1990

Expression of the epidermal growth factor gene in mouse lachrymal gland: comparison with that in the submandibular gland and kidney.

Expression of the epidermal growth factor (EGF) gene in mouse lachrymal gland was studied. The lachrymal gland of adult male and female mice contained immunoreactive EGF at a concentration of 0.013 +/- 0.002 (S.E.M.) and 0.014 +/- 0.003 ng/mg wet tissue respectively. Northern blot analysis of RNA from the lachrymal gland revealed the presence of 4.1-4.3 kb preproEGF mRNA, which was smaller in size than the 4.7-4.8 kb preproEGF mRNA in the submandibular gland and kidney. There was no sex difference in the mRNA level in the lachrymal gland, whereas the mRNA levels in the submandibular gland and kidney of adult male mice were 42-fold higher and twofold lower respectively than those of female animals. Administration of testosterone propionate or L-thyroxine to female mice increased submandibular gland levels of preproEGF mRNA by 35-fold or 12-fold respectively, but caused no change in lachrymal gland levels of preproEGF mRNA. The level of mRNA in the kidney was decreased by administration of androgen to one-third of that in control animals. The mRNA in the lachrymal gland was detected as early as 2 weeks of age and thereafter it remained at a constant level throughout adulthood, while that in the submandibular gland increased greatly during the pubertal stage. These results indicate differential hormonal and developmental regulation of EGF gene expression in the lachrymal gland, submandibular gland and kidney.

Animals↗
PubMed1989

Epidermal growth factor deficiency associated with diabetes mellitus.

The production of epidermal growth factor (EGF) in the submandibular gland and its circulating level were studied in diabetic mice. In genetically diabetic (C57BL/KsJ db/db) mice, EGF concentrations in the submandibular gland and plasma were reduced to 13% and 30% of the control levels, respectively. In streptozotocin-treated diabetic mice, they were reduced to 18% and 20% of controls, respectively, 5 weeks after the drug injection. Furthermore, levels of submandibular prepro-EGF mRNA in these diabetic mice were decreased almost in parallel with the glandular EGF concentrations, while there was no change in the levels of submandibular beta-actin mRNA and kidney prepro-EGF mRNA. In addition, histological examination of the submandibular glands indicated that the size of the granular convoluted tubules, which produce EGF, was substantially reduced in the diabetic mice. Insulin administration to streptozotocin-treated mice almost completely reversed the decrease in EGF content in the submandibular gland, substantially elevated the level of the glandular prepro-EGF mRNA and plasma EGF concentration, and increased the size of the granular convoluted tubules in the gland. These results indicate that EGF deficiency occurs in diabetes mellitus and that insulin may be important in maintaining the normal level of EGF in the submandibular gland and plasma.

Animals↗
PubMed1989

Impaired production of nerve growth factor in the submandibular gland of diabetic mice.

The production of nerve growth factor (NGF) in submandibular glands was examined in two kinds of diabetic mice. In genetically diabetic (C57BL/KsJ db/db) mice, which manifest marked insulin resistance and hyperglycemia, the concentration of NGF in the submandibular gland was less than one-tenth that of the nondiabetic controls. In streptozotocin-induced diabetic C57BL/KsJ mice, which show pancreatic insulitis leading to insulin deficiency and hyperglycemia, the glandular NGF concentration fell in a time-dependent manner to 26% of control level at 5 wk after the streptozotocin injection. A daily administration of insulin to the streptozotocin-induced diabetic mice restored the NGF concentration to almost the control level. The molecular size of NGF (13 kDa) in the glandular extracts of the genetically diabetic (db/db) mice in Western blots was indistinguishable from that of the control mice, but its level was reduced in the glands of the diabetic (db/db) animals. Although plasma NGF concentrations were normally below the sensitivity of the assay (less than 0.80 ng/ml) in both the control and the diabetic (db/db) mice, administration of cyclocytidine, which stimulates NGF release from the submandibular gland into the blood circulation, increased the plasma NGF level to 5.95 ng/ml in the control mice, but it failed to do so in the diabetic (db/db) mice. These findings suggest that, in diabetic mice, NGF production in the submandibular gland and its capacity to release NGF into the circulation are decreased.

Animals↗
PubMed1989

Decreased expression of hepatic epidermal growth factor receptor gene in diabetic mice.

The expression of hepatic epidermal growth factor (EGF) receptor gene was studied in genetically diabetic (C57BL/KsJ db/db) mice and streptozotocin-induced diabetic mice. The binding of 125I-labelled EGF to hepatic membrane preparations of genetically diabetic mice was only 35% of that of non-diabetic mice. Levels of EGF receptor messenger RNAs (10 and 6 kb) in the liver of the diabetic animals were also reduced by about 75%. In streptozotocin-induced diabetic mice, levels of hepatic EGF binding and messenger RNAs for EGF receptor were decreased to 27 and 30% of control levels respectively, at 5 weeks after injection of the drug. There were, however, no significant differences in levels of messenger RNAs for the structural protein beta-actin in the liver. In addition, levels of EGF receptor messenger RNAs in the kidney were similar between control and the two kinds of diabetic mice. Daily administration of insulin to the streptozotocin-induced diabetic mice increased the hepatic levels of EGF receptor messenger RNAs to almost normal levels. These results indicate that EGF binding to its receptor decreases in the liver of diabetic mice, involving alterations in the level of EGF receptor messenger RNAs, and that insulin is important for the regulation of EGF receptor gene expression in the liver but not in the kidney.

Actins↗
PubMed1989

The regulation by thyroid hormones and androgen of epidermal growth factor synthesis in the submandibular gland and its plasma concentrations in mice.

The effects of androgen and thyroid hormones on epidermal growth factor (EGF) synthesis in the submandibular gland and on plasma EGF concentrations in mice were examined. Testosterone propionate was administered alone or in combination with L-thyroxine (T4) to female mice for 2 weeks. The submandibular EGF concentrations were increased by the administration of testosterone propionate in a dose-dependent fashion; the maximal increase, 20-fold, being produced by a dose of 2 mg every other day. The EGF levels were increased sevenfold by T4, which also enhanced the stimulatory effect of suboptimal doses of testosterone propionate. Cyproterone acetate, an anti-androgen, inhibited the testosterone propionate-induced increase, but not the T4-induced increase. Plasma EGF concentrations were raised by testosterone propionate but not by T4. Both hormones stimulated the accumulation of 4.7 kb preproEGF mRNA in the submandibular gland, which occurred almost in a parallel manner with the increase in submandibular EGF concentrations. These results suggest that EGF synthesis in the submandibular gland is regulated by alterations in the level of its mRNA by thyroid hormones and androgen, and that the rise in plasma EGF concentrations is under the influence of androgen but not of thyroid hormones.

Animals↗
PubMed1987

Sodium molybdate converts the RNA-associated transformed, oligomeric form of the glucocorticoid receptor into the transformed, monomeric form.

The glucocorticoid receptor from rat liver cytosol prepared in 2 ml buffer/g tissue sedimented at approximately 10 S in low salt density gradient centrifugation without molybdate. When the receptor was heated at 25 degrees C, both approximately 10 S and approximately 7 S forms were seen in low salt gradient. The approximately 10 S form was not capable of binding to DNA-cellulose and was stabilized by sodium molybdate, namely it corresponded to untransformed receptor. The approximately 7 S form was capable of binding to DNA-cellulose and regarded as transformed receptor. On the other hand, partially-purified transformed receptor labeled with [3H]dexamethasone-21-mesylate sedimented at approximately 5 S, which migrated as a approximately 94 kDa species in SDS-polyacrylamide gel electrophoresis. The reconstitution analysis of this partially-purified approximately 5 S receptor and liver cytosol, showed the shift to approximately 7 S form. RNase A or T1 converted approximately 7 S transformed form into approximately 5 S but it did not affect approximately 10 S untransformed form. 5-20 mM sodium molybdate also shifted approximately 7 S to approximately 5 S. These results indicate that the approximately 7 S transformed form of the glucocorticoid receptor observed in low salt conditions might be an oligomer, probably including both approximately 5 S steroid-binding component and RNA/ribonucleoprotein, and that molybdate dissociates these interactions in a specific manner.

Adrenalectomy↗
PubMed1987

Leupeptin inhibits the transformation of glucocorticoid receptor.

The effect of leupeptin upon the transformation of the glucocorticoid receptor was tested. When the labeled receptor was treated with heat or high salt in the presence of leupeptin, the binding to DNA-cellulose decreased in a dose-dependent manner. We observed 50% inhibition with about 40 mM leupeptin. The addition of leupeptin after the transformation procedures did not inhibit the binding to DNA-cellulose. In gradient centrifugation, 40 mM leupeptin retained approximately 10S, untransformed form. Elution profiles from DEAE-cellulose showed the preservation of the peak eluted with 0.2 M KCl, corresponding to the untransformed form. These results indicate that leupeptin might have the similar effects to molybdate in regard to blocking the transformation of rat liver glucocorticoid receptor, though the effects with leupeptin were not as great as those seen with molybdate.

Adrenalectomy↗