Biomedical subjects

H Kouhara

Publications and source records attributed to H Kouhara.

32 records · Page 2Linked to original sources

PubMed1994

Transforming activity of a newly cloned androgen-induced growth factor.

Our previous study demonstrated that androgen-dependent growth of mouse mammary carcinoma cells (SC-3) was mediated through an induction of heparin-binding growth factor, termed as androgen-induced growth factor (AIGF). Here, we report that NIH3T3 cells stably transfected with AIGF expression vector exhibit the abilities of tumor formation in nude mice, focus formation in monolayer culture and colony formation in soft agar. Thus, this newly cloned growth factor can be categorized as an oncogene. In addition, androgen-induced enhancement of DNA synthesis in SC-3 cells can be blocked by simultaneous incubation with AIGF antisense oligonucleotides. The possibility is also addressed that AIGF exerts its biological activity through an interaction with fibroblast growth factor (FGF) receptor. Transfection of expression vector encoding a variant form of FGF receptor-1 cloned from SC-3 cells into FGF receptor-negative L6 cells results in AIGF-dependent inhibition of differentiation. These results demonstrate that the ability of androgen to elicit transformed phenotype of SC-3 cells is mediated through AIGF induction and its interaction with FGF receptor-1.

Animals↗

PubMed1993

Suramin interrupts androgen-inducible autocrine loop involving heparin binding growth factor in mouse mammary cancer (Shionogi carcinoma 115) cells.

The androgen-dependent clonal cell line SC-3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)-like autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC-3 cells, FGF receptor expression is upregulated by the SC-3-derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effects of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC-3 cells were studied. Suramin inhibited androgen-dependent growth of SC-3 cells in a concentration-dependent fashion: approximately 50% inhibition was observed at 25 microM. [3H]Thymidine incorporation into the cells stimulated with partially purified SC-3-derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF-induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC-3 cells with suramin decreased cell surface 125I-bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 microM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor-1 mRNA to a similar extent, whereas it appeared not to affect the levels of beta-actin mRNA. Moreover, suramin completely blocked androgen- or bFGF-induced accumulation of FGF receptor-1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC-3 cells through inhibition of an androgen-inducible autocrine loop involving SC-3-derived growth factor and FGF receptor.

Animals↗

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

[Molecular mechanism of androgen-dependent transformation--analysis using serum-free culture system].

SC-3 cells, derived from androgen-dependent mouse breast cancer Shionogi carcinoma 115, have shown not only androgen-dependent growth stimulation but also morphological changes in serum-free culture. We have demonstrated that this androgen-dependent growth is mediated through an induction of heparin-binding growth factor (termed as androgen-induced growth factor: AIGF). We studied the molecular mechanism of androgen-dependent growth. NIH3T3 cells transfected to express AIGF showed focus-forming activity, anchorage-independent growth and tumor formation in nude mice indicating AIGF is an oncogene. The findings that AIGF inhibited binding of [125I]basic fibroblast growth factor (bFGF) and only FGF receptor (FGFR)-1 mRNA expressed in SC-3 cells suggested that AIGF acts through FGFR-1. Therefore, we cloned FGFR-1 from cDNA library of SC-3 cells, which was found to have three mutations. When this variant FGFR-1 was expressed in FGFR-negative myoblast L6 cells, AIGF-dependent inhibition of the differentiation to myocytes was observed suggesting that variant FGFR-1 is receptor of AIGF. Since expressions of c-myc mRNA as well as AIGF mRNA were induced by testosterone treatment, we studied the role of c-myc in androgen-dependent growth in SC-3 cells using phosphorothioate derived antisense oligonucleotides (antisense) of c-myc or AIGF. Not only AIGF antisense but also c-myc antisense inhibited androgen-dependent growth. c-myc antisense did not affect androgen-induced AIGF mRNA expression. c-myc antisense inhibited AIGF-, but not bFGF-induced DNA synthesis. These results suggest that c-myc has a critical role of AIGF-, but not bFGF-induced growth.

3T3 Cells↗

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

Intracranial and intraspinal dissemination of an ACTH-secreting pituitary tumor.

A case of a 29-year-old man with an ACTH-producing pituitary tumor disseminated into the subarachnoid space is described. After total adrenalectomy for Cushing's disease at the age of 15, Nelson's syndrome developed. Transsphenoidal adenomectomy at 17 and 21 years of age, pituitary irradiation and medical therapies with sodium valproate, baclofen and bromocriptine failed to lower his plasma ACTH level. Multiple intracranial and intraspinal tumors associated with the symptoms of left hemiparesis developed. The removal of a tumor grown at the level of C1-3 was performed with successful palliation of his symptoms. Histologically, the tumor cells showed sinusoidal, papillary and diffuse patterns with a preponderance of the former over the latter two, although the papillary pattern predominated in the primary pituitary tumor. Immunohistochemical analysis demonstrated most cells to be positive for ACTH in the metastatic tumor as well as the primary adenoma. The clinical significance of his course is discussed with a review of 11 reported cases with metastatic ACTH-producing pituitary tumors.

Adrenocorticotropic Hormone↗

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

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

Effect of retinoic acid on proliferation of estrogen-responsive transformed murine Leydig cells in serum-free culture.

B-1 F cells, one of the sublines established from mouse Leydig cell tumor, have been found to be maintained as an estrogen-responsive cell line under the serum-free culture conditions. Reported results that retinoids have action mechanisms similar to those of estrogen prompted us to examine the effect of retinoids on the proliferation of B-1 F cells. Stimulation of B-1 F cell growth by retinoic acid in a dose-dependent manner was observed, whereas retinoic acid did not promote but inhibited the proliferation of MCF-7 cells (estrogen- and retinoic acid receptor-positive human breast cancer cells). To elucidate the mechanism of retinoic acid-dependent cell growth, simultaneous treatment with retinoic acid and estradiol was carried out. The result did not show the additive effect on B-1 F cell growth. Hydroxytamoxifen, a potent antiestrogen, inhibited not only estradiol-dependent but also retinoic acid-dependent cell growth. However, retinoic acid failed to be associated with estrogen receptor, suggesting that retinoic acid induced enhancement of B-1 cell growth through its interaction with retinoic acid receptor. Northern blot analyses of polyadenylated RNA with complementary DNA probes for human retinoic acid receptor alpha, beta, and gamma revealed the presence of transcripts encoded by retinoic acid receptor alpha gene in B-1 F cells. These results would suggest that enhancement of the B-1 F cell growth is mediated through interaction of retinoic acid with retinoic acid receptor alpha. This stimulatory activity is inhibited by estrogen receptor complexed with hydroxytamoxifen.

Animals↗

PubMed1990

Identification of unoccupied but transformed nuclear estrogen receptor in cultured mouse Leydig cell.

The molecular forms of estrogen receptor (ER) in estrogen-responsive mouse Leydig cell line (B-1) have been examined in relation to their biological activity. ER was predominantly recovered in the nuclear fraction upon homogenization even after cells were precultured in the absence of E2 and Phenol Red. This unoccupied nuclear ER (ERn) whose hormone binding ability was extremely thermostable could be extracted with 0.4 M KCl. This stability enabled us to determine hydrodynamic parameters in the ligand-free condition. The Stokes radius and sedimentation constant of this ERn in high salt condition were 5.5 nm and 6.0S, respectively, resulting in its molecular weight of 140,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ER labeled with [3H]tamoxifen aziridine gave a single band of 65,000 Da, indicating that this ERn had a oligomer structure similar to that of transformed nuclear ER complexed with estrogen in the putative target cells. Therefore, we further examined the possibility that this ERn in B-1 cells can activate estrogen-responsive genes without any aid from estrogen. Estrogen responsive element-thymidine kinase promoter-chloramphenicol acetyltransferase fusion gene (ERE-tk-CAT) was transfected into B-1 cells. CAT activity was enhanced only in cells stimulated with estrogen. It may be concluded from these results that transformed ERn can be formed in the absence of estrogen but that binding to estrogen may be required in order to exert its biological activity.

Animals↗

PubMed1987

Direction of transcription affects the replication mode of lambda in an in vitro system.

A set of artificial circular plasmids, named plasmoids, was constructed. They are about 1 kb in size and consist of a 178 bp lambda dv minimal DNA replication origin (ori) which has four direct repeats and the A + T-rich region conferring polarity to the ori fragment, a 775 bp DNA segment that codes for the CAT amino acid sequence and the 99 bp lac promoter (plac). They carry no other functional genes or genetic sites. The constructions involved various combinations of relative orientations of these components. These molecules do not replicate in vivo because they lack genes coding for initiation proteins, but they do replicate in an in vitro system (Fuller et al. 1981), and can be used for studies of interactions between transcription and replication. In these plasmids, major transcription starts from the strong plac, and some weak unscheduled transcription starts from several other initiation sites. The major RNA synthesis was found to interfere with the unscheduled RNA synthesis, which was occurring on the opposite strand. The most active replication took place when the major RNA synthesis went through the lambda origin region in the direction which occurs naturally in the lambda genome. Under these conditions, DNA synthesis going against such transcription was less than that going along with the major transcription. When RNA synthesis through the lambda origin region was in the opposite direction, DNA synthesis in the same direction was about half of that observed in the above case, whereas that going against transcription was very weak.

Bacteriophage lambda↗

PubMed1987

[Regulatory mechanism of steroid hormone receptor functions].

The steroid hormone receptor has been defined as an intracellular protein which associates with the corresponding ligand in a stereospecific manner. Its binding ability and affinity have been reported to be affected by many factors such as protein kinase, SH reducing agent and molybdate. Through ligand binding, hormone-receptor complexes undergo activation process resulting in its acquisition of high affinity toward DNA. Although the detailed biochemical mechanism of receptor activation remains to be elucidated, the reduction of their molecular size has been proposed to be an obligatory step for receptor activation. Recently, heat shock protein 90 K, has become known to bind with the oncogene product (pp 60 v-src). Furthermore, it has been demonstrated to be a common component of both nonactivated, and not activated steroid receptor. The recombinant gene technology has successfully determined the amino acid sequence of GR, ER and PgR. All steroid receptors consist of three domains, namely the immunogenic, DNA-binding and steroid-binding domains. Experiments using deletion mutant receptors revealed that the DNA binding domain has both abilities of DNA binding and transactivation, and that the steroid binding domain negatively regulates DNA binding activity. The purified GR has been found to bind with the specific region (GRE) of glucocorticoid-dependent genes. The consensus sequence of GRE has been observed to be TG-TTCT. Two other proteins which can be associated with some regions near GRE have been proposed to activate cooperatively the glucocorticoid responsive genes with GR. Therefore, the steroid hormone receptor systems are suitable for clarifying the molecular mechanism of gene activation.

Animals↗

PubMed1985

Origin and initiation sites of lambda dv DNA replication in vitro.

The sequence of lambda DNA essential for the unique initiation of replication was analyzed in an in vitro replication system. Fragments of lambda DNA were inserted into pBR322 and used as templates or were circularized in vitro in the absence of pBR322 and employed in the same tests. A 165-bp region left of the EcoRI site in the O gene of the lambda genome was defined as the functional origin. This region, which we defined as the ori region, carries, in order, the 4 19-bp repeat sequences where the O protein binds (ori-repeats), an A+T-rich stretch, and a region that constitutes part of a large palindromic structure. Regions that have long been suspected to participate in lambda DNA replication initiation, ice and oop were not required for the O, P-dependent lambda-specific replication initiation. The lambda dv and the "ori region plus" recombinant plasmids initiated DNA synthesis at or around this region, and the reaction depended on the presence of lambda-coded initiators, O and P proteins. Early replicative intermediates of lambda dv were prepared in an in vitro replication system in the presence of ddCTP, an inhibitor of DNA chain elongation. This system allows only that DNA synthesis that is a result of replication initiation. Using this system, the initiation site(s) of the DNA synthesis was finely analyzed by mapping the transition sites from primer RNA to DNA synthesis. Short-chain DNAs produced from regions near the ori region were purified from the intermediates. A fraction of the short-chain DNAs was covalently linked to primer RNA. The 5'-ends that had been linked to RNA (transition sites) were exposed by alkaline hydrolysis, labeled with 32P, and the transition sites were mapped along the nucleotide sequence of the genome. Two striking features emerged from this analysis: (i) The transition sites are located on both sides of the ori region, and no transition arose within the 165-bp ori region; (ii) The transition sites on both sides are not unique, but multiple, and are clustered in one of the 2 strands. Furthermore, their orientation demonstrates that the DNA synthesis in initiation of replication from the 2 sides of the ori region converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis. These results reflect asymmetric bidirectional replication of lambda dv DNA, and may also reflect replication of lambda phage DNA.

Bacteriophage lambda↗