Bunzo Sato

Advanced glycation endproducts stimulate interleukin-6 production by human bone-derived cells

Advanced glycation endproducts (AGEs), which result from nonenzymatic reactions of glucose with tissue proteins, have been shown to accumulate on long‐lived proteins in advanced aging and diabetes mellitus. Thus, AGEs have been implicated in some of the chronic complications associated with these disorders. In this study, we investigated the effects of the glucose‐modified protein on the production of the potent bone resorption factors by cells derived from explants of human bone. AGEs stimulated the release of interleukin‐6 (IL‐6) in the culture supernatants from the bone‐derived cells and increased the levels of IL‐6 mRNA in the cells. By contrast, the levels of IL‐11 in the culture supernatants were not altered by AGEs, and the other bone resorption factors IL‐1α and IL‐1β were undetectable (<1.0 pg/ml) either without or with the treatment of AGEs. Electrophoretic mobility‐shift assays revealed that the transcription nuclear factor‐κB, which is critical for the inducible expression of IL‐6, was activated in the nuclear extracts from mouse osteoblastic MC3T3‐E1 cells treated with AGEs. These results suggest that AGEs are involved in bone remodeling modulation by stimulating IL‐6 production in human bone‐derived cells.

Functional and possible physical association of scavenger receptor with cytoplasmic tyrosine kinase Lyn in monocytic THP-1-derived macrophages

Acetyl LDL (modified low‐density lipoprotein), which is thought to be taken up through scavenger receptor A (SR‐A), rapidly induced the appearance of phosphotyrosine proteins in monocytic THP‐1‐derived macrophages in vitro. The two alternative forms of Lyn (p53 and p56) were found to be tyrosine‐phosphorylated within 30 s after the stimulation with acetyl LDL. The catalytic activity of Lyn measured by an in vitro kinase assay had also increased in acetyl LDL‐stimulated THP‐1‐derived macrophages. Furthermore, Lyn could be co‐immunoprecipitated with SR‐A from the cell lysate. These observations suggest a functional and possible physical association of SR‐A with Lyn in THP‐1‐derived macrophages, and also imply a possible involvement of Lyn in SR‐A signal transduction.

Potent novel nonsteroidal androgen antagonists with a phthalimide skeleton

Abstract Anti-androgenic activity of various phthalimide analogs was evaluated based on inhibition of androgen-induced activation of nuclear androgen receptor (CAT assay) and on growth inhibition of the androgen-dependent clonal cell line SC-3. Some compounds showed very potent androgen-antagonistic activity.

Steroid receptors in osteoblasts.

Using the whole-cell incubation system at 37 degrees C, the specific bindings for 3H-dexamethasone, 3H-estradiol-17 beta, 3H-dihydrotestosterone and 3H-R5020 were measured in the purified, putative osteoblasts isolated from fetal rat calvaria by collagenase digestion. More than 90% of the purified cells contained intense alkaline phosphatase activity. The specific binding for 3H-dexamethasone with high affinity and low capacity was demonstrated in the isolated osteoblasts. Most of the binding was found in the nuclear fraction, indicating nucler binding of the 3H-dexamethasone-receptor complex. The apparent dissociation constant (Kd) for 3H-dexamethasone was estimated to be 3.3 x 10(-9)M and the number of binding sites was calculated to be 65 fmol/ml (4 x 10(6) cells) or 9,750 binding sites per cell. High salt: sucrose gradient analysis of nuclear extracts revealed a radioactive 4.0 S peak. These results indicate that the purified osteoblasts are among the target cells for glucocorticoids. On the other hand, the specific bindings for 3H-estradiol-17 beta and 3H-dihydrotestosterone were not detectable in the isolated osteoblasts, which suggests that estrogens and androgens act on osteoblasts only indirectly.

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.

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.

Hormone Receptor in Renal Cell Carcinoma and Correlation With Clinical Response to Endocrine Therapy

Analyses of hormone receptors in cytosols from 41 renal cell carcinoma specimens were performed by the dextran-coated charcoal technique, using estradiol, synthetic progestin R5020 and synthetic androgen R1881. Binding data were calculated according to the method of Scatchard. Of 41 renal cell carcinomas estradiol receptor was detected in 11, R5020 receptor in 11 and R1881 receptor in 13. No significant correlation between histopathological findings and hormone receptors was observed. Patients were classified into those positive and negative for receptors. The clinical response of endocrine therapy for 17 with advanced residual or metastatic lesions after nephrectomy was studied in regard to the survival rates. Although there was no complete or partial regression in tumor size, the survival rate of patients with 1 or more receptors was significantly higher than that of patients negative for receptors (p less than 0.01). In conclusion, hormonal manipulation in patients with renal cell carcinoma cannot induce an antitumor effect but seems to increase survival in patients with receptors.

Hormonal regulation of activities of 17β-ol-dehydrogenases, aromatase and 4-ene-5α-reductase in immature rat ovaries

Abstract Female rats were hypophysectomized at 21 days of age, and after 3 days, the hypophysectomized rats in groups of 3–20 were injected daily with 10 μg of NIH-LH-S19, 10–100 μg of NIAMD-Rat-FSH-B-1, 20 μg of oestradiol-17β or saline for 3 days. Ovarian homogenates from these rats and intact rats at 27 days of age were incubated with [14C]-4-androstene-3,17-dione, [14C]-oestrone, [7-3H]-4-androstene-3,17-dione or [7-3H]-progesterone and enzyme activities and metabolism of progesterone were estimated. The activities of 5α-reductase, testosterone and oestradiol 17β-ol-dehydrogenases and aromatase decreased significantly 6 days following hypophysectomy. A distinct response to LH but not to FSH in the 5α-reductase activity in the hypophysectomized rat ovary was found. On the other hand, the activities of 17β-ol-dehydrogenases and aromatase in the hypophysectomized rat ovary were stimulated (10 to 200 times) by FSH but not by LH. No stimulation of these enzyme activities by oestradiol- 17β was involved. The formation of oestradiol-17β from progesterone could be demonstrated only in the FSH-injected rat ovary. These results show that the 5α-reductase activity is regulated by LH and the activities of 17β-oldehydrogenases and aromatase are regulated by FSH in immature rat ovaries. It is also suggested that nonresponse to LH and response to FSH of the uterus of hypophysectomized immature rats can be explained in part by the present results.

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.

Growth-stimulatory effects of androgen, high concentration of glucocorticoid or fibroblast growth factors on a cloned cell line from Shionogi carcinoma 115 cells in a serum-free medium

The effects of various kinds of growth factors or steroids on the proliferation of Shionogi carcinoma 115 (SC115) cells were investigated in cell culture. In a serum-free medium [Hams F-12:Eagles minimum essential medium (1:1, vol/vol) containing 0.1% bovine serum albumin], the proliferation of SC-3 cells (a cloned cell line from SC115 cells) estimated by [3H]thymidine incorporation into DNA and cell number reached a plateau at 10(-8) M testosterone (up to 200-fold), 10(-7) M dexamethasone (up to 30-fold) or 1 ng/ml of fibroblast growth factors (FGF; up to 50-fold). However, the proliferation in the serum-free medium was not significantly stimulated by the addition of low to very high concentrations of progesterone, oestradiol-17 beta, epidermal growth factor, platelet derived growth factor or insulin; transforming growth factor beta slightly stimulated the growth (up to 5-fold) but markedly inhibited the growth stimulation induced by testosterone. Furthermore, an epithelial appearance of SC-3 cells grown in the absence of growth factors or steroids was changed to a fibroblast-like appearance only by the addition of testosterone, high concentrations of dexamethasone or FGF. By investigating various kinds of growth factors or steroids, the present study demonstrates that androgen, high concentration of glucocorticoid or FGF alone significantly stimulates the proliferation of SC-3 cells with a change of morphology in the serum-free medium.