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1.
B Sato 《Human cell》1989,2(3):246-253
Steroid hormone-responsive cell lines were clones from mouse mammary cancer (Shionogi Carcinoma 115) and Leydig cell tumor. SC-3 and SC-4 cells from Shionogi Carcinoma were androgen-responsive and -unresponsive in a serum-free medium, respectively. SC-3 cells secreted FGF-like growth factor as well as 24 K glycoprotein in response to androgen stimuli. B-1 and B-1F cells from mouse Leydig cell tumor were growth-stimulated in a serum-free medium by estrogen, androgen or retinoic acid. Transfection of ERE-TK-CAT gene into B-1F cells revealed that both estrogen and retinoic acid activated the CAT activity. In addition, the presence of corresponding receptors for steroid hormones or retinoic acid was demonstrated by hormone binding assays and/or Northern blot analysis. Thus, these serum-free culture systems seem to be very useful for analysing hormone action mechanisms in vitro.  相似文献   

2.
Shionogi carcinoma 115 (SC115) had been accepted for 20 yr as an androgen-dependent mouse mammary tumor. However, we recently found that the growth of SC115 tumors in vivo is also stimulated by pharmacological doses of estrogen through estrogen receptor. In the present study, action mechanisms of androgen or high doses of estrogen in the growth stimulation of SC115 were examined using a cloned cell line (SC-3) derived from the SC115 tumor. In serum-supplemented [2% steroid-free fetal calf serum-Eagle's minimum essential medium (MEM)] and serum-free [HAM F-12: MEM (1:1, v/v) containing 0.1% bovine serum albumin] media, testosterone (Test, 10(-9)-10(-6) M) significantly increased both cell number and DNA synthesis of SC-3 cells (by up to 10-fold), whereas oestradiol-17 beta (10(-12)-10(-6) M) had no such effects; the Test-induced growth was completely inhibited by the addition of a 100-fold molar excess of cyproterone acetate (CA). The serum-free medium cultured with SC-3 cells in the presence or absence of 10(-8) M Test was collected [conditioned medium (CM) or conditioned medium without Test (CM-)], and then Test in CM was removed by Gel filtration using Sephadex G-100 or inactivated by the addition of a 100-fold molar excess of CA. In the serum-free culture system, the addition of the CM without Test activity significantly enhanced both number of SC-3 cells and DNA synthesis in the cells, whereas CM(-) had no such effects. The present findings suggest that growth-stimulatory activities of androgen and high doses of estrogen on SC115 cells are mediated by growth factor(s), secreted from SC115 cells through androgen receptor and from some of nontransformed cells through estrogen receptor, respectively.  相似文献   

3.
Upon androgen deprivation, Shionogi (SC-115) mouse mammary tumors undergo phenotypic changes enabling their escape from growth dependence on androgens. Even within androgen-responsive cell populations, marked clonal heterogeneity is observed in the trophic effects of androgens. The present study compares several parameters of androgen action between three SC-115 cell clonal subpopulations exhibiting high (clone 107), low (clone S1A2) and no trophic response (clone 415) to androgens. These parameters pertain to (1) kinetics of androgen binding, (2) metabolism of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol), (3) ornithine decarboxylase (ODC) activity and (4) interleukin-1alpha (IL-1alpha) action on cell proliferation. Only marginal differences in the affinity and abundance of androgen-specific binding sites were detected between the three clones. While clone S1A2 degraded DHT to 3alpha-diol at a much faster rate than the highly androgen-sensitive 107 cells and androgen-insensitive 415 cells, differences in the rates of intracrine conversion of 3alpha-diol and 3beta-diol to DHT did not correlate with the ability of these steroids to stimulate cell proliferation. Induction of ODC activity at the onset of exponential growth was strongly DHT-dependent in 107 cells, whereas this dependence was markedly attenuated in androgen-hyposensitive cells. Unexpectedly, DHT strongly repressed the marked ODC induction resulting from fresh medium addition in 415 cells which show no growth response to androgens. Low IL-1alpha concentrations were mitogenic in all three SC-115 clones. Whereas the mitogenic action of IL-1alpha was completely androgen-dependent in 107 cells, this dependence was relieved in S1A2 cells, which responded to DHT and IL-1alpha in an additive fashion. Thus, clonal heterogeneity in the pattern of steroid metabolism within Shionogi tumors cannot solely account for loss of androgen dependence, which may rather correlate with the constitutive activation of transduction pathways controlling the expression of growth-associated genes (e.g. ODC) by serum growth factors, including IL-1alpha.  相似文献   

4.
A retroviral insertional mutation, especially by mouse mammary tumor virus (MMTV), is a major cause of murine mammary tumorigenesis. Prompted by our previous finding that FGF8, an insertionally activated cellular oncogene, is highly expressed in androgen-dependent mouse mammary Shionogi carcinoma cells, we here investigated retroviral integration adjacent to the fgf8 locus in Shionogi carcinoma. In the genomic Southern blots for fgf8 and its 5'-upstream gene npm3, the hybridized fragments were identical to the host DD/Sio mice, the original Shionogi carcinoma 115 tumor, and a pair of cultured Shionogi carcinoma cell lines of SC-3 and SC-4, suggesting that no retroviral integration occurred around either loci. The genomic cloning for the fgf8 locus from SC-3 cells also confirmed no MMTV integration. In addition, npm3, which is usually coactivated with fgf8 by MMTV insertion,was not up-regulated by androgens in SC-3 cells. All these findings led us to conclude that no retroviral insertion was present at the common integration sites adjacent to the fgf8 locus in Shionogi carcinoma although we demonstrated in this study that multiple proviral sequences of MMTV, Moloney murine sarcoma virus and FBJ-murine sarcoma virus are integrated into SC-3 cells in association with their distinct promoter activity in SC-3 cells.  相似文献   

5.
Cancer of the prostate is the most frequent cancer and the second leading cause of cancer death in men in North America. The growth of Shionogi carcinoma-115 (SC-115) cells is highly sensitive to androgens, and this cell line is a well known experimental model of prostate cancer. The transplantable Shionogi carcinoma tumor was used to assess the influence of tumor size on the response to flutamide treatment. Two weeks after subcutaneous inoculation of tumor fragments in Shionogi mice, six groups of animals bearing SC-115 tumors ranging from 0.1 to 1.8 cm in diameter were treated with flutamide (1 mg, twice daily). The castrated mice received an androstenedione (Δ4-dione) implant to mimic the human situation, where the adrenals produce precursor steroids which are transformed into androgens in peripheral intracrine tissues. After 16 days, treatment with flutamide inhibited tumor growth by 32 to 57% in the four groups of mice having tumors ranging from 0.1 to 1.0 cm in diameter at day 0, whereas no significant inhibitory effect was observed in larger tumors. The same treatment, however, caused potent inhibitory effects on other androgen-sensitive parameters, namely prostatic and seminal vesicle weight and kidney ornithine decarboxylase (ODC) activity, the effect on these parameters being similar in all groups of animals, irrespective of tumor size. Furthermore, when those larger tumors unresponsive to antiandrogenic treatment were cut into small fragments and inoculated into new groups of mice, the same treatment with flutamide efficiently inhibited tumor growth, treatment being started at tumor sizes of 0.1 to 0.3 cm in diameter. The present data clearly demonstrate that small tumors are highly sensitive to androgen deprivation, while loss of response develops with increasing tumor size, thus indicating that, for optimal efficacy, androgen blockade should be given at the early stages of prostate cancer.  相似文献   

6.
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 [Ham's F-12:Eagle's 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.  相似文献   

7.
Antiglucocorticoid and antiprogestin RU38486 (RU486) stimulated the growth of highly androgen- and moderately glucocorticoid-sensitive SC-3 cells (a cloned cell line from Shionogi mouse mammary carcinoma 115) in a dose-dependent manner. A maximal 8-fold stimulation of growth by RU486 has been observed at 10(-7) M in a serum-free medium and its potency has been found to be almost the same as that of dexamethasone (Dex). The growth rate of SC-3 cells treated by triamcinolone acetonide (TA) or Dex combined with RU486 at 10(-9)-10(-7) M was enhanced compared to cells treated by TA or Dex alone, indicating that RU486 had additive rather than antagonistic effects. Our previous study revealed that RU486 could compete with the specific uptake of [3H]testosterone in intact SC-3 cells at relatively low affinity and the present study showed that the stimulatory effect of RU486 on the growth of SC-3 cells was significantly inhibited by pure antiandrogen flutamine and that half-maximal inhibition by flutamine was achieved at 10(-6) M. Moreover, we demonstrated that the conditioned medium from RU486-stimulated SC-3 cells contained growth-promoting activity which caused a 3.5-fold increase in DNA synthesis by SC-3 cells in the absence of RU486 and which was abolished by treatment with heparin-Sepharose. These results indicate that RU486-induced growth of SC-3 cells may be expressed as an androgenic activity through androgen receptor and mediated by a heparin-binding growth factor.  相似文献   

8.
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.  相似文献   

9.
The androgen-dependent clonal cell line SC-3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)-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 effect 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: ~50% inhibition was observed at 25 μM. [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 μM 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 β-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. © 1993 Wiley-Liss, Inc.  相似文献   

10.
Primary cell cultures derived from an androgen-dependent mouse mammary carcinoma, the Shionogi SC-115 tumor, display characteristic changes in growth, morphology and protein synthesis according to the presence or absence of testosterone. In the presence of testosterone, cell proliferation was increased and cells formed characteristic clones having no contact inhibition. Ultrastructural studies of cells showed close contacts of plasma membranes having little or no gap between cells. Some cells were related by bridges of extracellular matrix. Testosterone-induced synthesis of several intracellular and secreted proteins was observed after [35S]methionine-labeling of cells, SDS-PAGE and autoradiography, as well as the disappearance of a protein in androgen-treated cells. In the absence of testosterone, cells grow as a monolayer, have contact inhibition and flattened morphology. The ultrastructurally observed cell-to-cell contacts were usually less intimate, showing spaces of irregular width between cells. None of the testosterone-induced proteins were observed in the absence of hormone. The antiandrogen cyproterone acetate, which by itself was inactive, completely suppressed the androgen-induced effects on growth, morphology and specific protein synthesis. Glycosylation of membrane proteins, as measured after labeling of cells with [3H]N-acetyl-D-glucosamine, was increased by approximately 30% in the presence of testosterone. A similar observation was made in situ by autoradiography on intact cells. Finally, we found that culture medium conditioned by testosterone-treated Shionogi cells had significant mitogenic activity on L-929 mouse fibroblasts.  相似文献   

11.
The mouse androgen dependent tumor Shionogi carcinoma 115 or the non-responsive tumor Shionogi carcinoma 42 were transplanted into 40 mice and testosterone-3H (0.13 μg/20 μCi) was then injected into each animal. The retention of ether soluble radioactivity in the nuclear, mitochondrial, microsomal and cytosol fractions was higher in combined seminal vesicle-prostate tissue and in Shionogi carcinoma 115 than in Shionogi carcinoma 42, muscle, and spleen.  相似文献   

12.
A radioautographical technique for the localization of soluble compounds in cultured cells is described. It has been used to investigate the distribution of steroid hormones in target cells, and an example is given with the Shionogi carcinoma SC-115, an androgen-sensitive mammary tumor in mice. Experiments at 37 °C have given direct evidence for the specific binding of [3H]-androstanolone (5α-dihydrotestosterone) in the cytoplasm. This result is based on the labeling after incubation with 0.5 nM radioactive androgen, on the isotope dilution-test showing a limited capacity, and on competition assays with other hormones. Results also show the transfer to and the specific binding of the hormone in the nucleus. No cellular labeling has been observed at 0 °C, even when using 10 nM of high specific activity [3H]androstanolone. The technique appears promising for the study of various aspects of the interactions between hormones and isolated cells.  相似文献   

13.
In order to study the regulation of aromatase activity by androgens in cultured fibroblasts derived from genital skin of normal prepubertal boys, aromatase activity was evaluated in the presence of various concentrations of non-aromatizable androgen DHT(5 alpha-dihydrotestosterone). The estrogen formation was assayed by an enzymatic method, after 24 h incubation of the cells with 10(-6) M androstenedione. Aromatase activity was stimulated 3- to 20-fold by DHT at concentrations 10(-10) and 10(-9) M. It was necessary to preincubate the cells with DHT for 48 h in order to bring about this stimulation. The stimulatory effect was not significant after preincubation for only 24 h. The basal value of aromatase activity was in the range of 8 +/- 1.2 pmol/mg protein/day (mean +/- SEM), while the maximal stimulation 1043 +/- 46 pmol/mg protein/day was obtained at the concentration of 10(-8) M DHT. This stimulation was partially blocked with cyproterone acetate at level of 20 +/- 4 pmol/mg protein/day; stimulation of aromatase activity by DHT could thus be mediated by the androgen receptor. This stimulatory effect was prevented by incubation of the cells with cycloheximide or actinomycin D, suggesting that DHT acts to increase aromatase activity in cultured fibroblasts by inducing the synthesis of new proteinaceous material. In vitro regulation of aromatase activity by androgens could contribute to a new approach to the extraglandular formation of estrogen.  相似文献   

14.
3-Substituted (Z)-4-(4-N,N-dialkylaminophenylmethylene)-5(4H)-isoxazolones and related compounds were designed and prepared as candidates for structurally novel androgen antagonists. Several compounds showed potent anti-androgenic activity as assessed by nuclear androgen receptor binding assay and growth inhibition assay using androgen-dependent Shionogi carcinoma cells SC-3. They were approximately 10--220 times more potent than flutamide in these assay systems. They also showed anti-androgenic activity toward prostate tumor cell line LNCaP, which has an aberrant nuclear androgen receptor.  相似文献   

15.
B A Ponder  L V Crawford 《Cell》1977,11(1):35-49
Androgen-dependent and androgen-independent (autonomous), cloned, cultured cell lines of the androgen-dependent mouse mammary adenocarcinoma, Shionogi carcinoma 115, have been established. Growth of the dependent cells requires the presence of androgen, provided they are grown in suspension culture in medium containing dextran-charcoal-treated fetal calf serum. The growth rate of autonomous cells in the presence or absence of DHT is similar to that of dependent cells grown in its presence. An agar culture method has been developed that enables the proportion of dependent and autonomous cells in mixed populations to be determined. Autonomous cells appear in dependent clones, and their frequency increases with increasing time of subculture. Dependent cells from tumors preferentially in male animals, and dependent cell cytosols contain significant amounts (approximately 300 femtomoles per mg protein) of a specific androgen-binding macromolecule. Autonomous cells formed tumors equally well in both male and female mice, and autonomous cell cytosols contain very low levels (≤ 7 femtomoles per mg protein) of the specific androgen-binding macromolecule(s). These studies delineate a system which can be used to investigate the mechanism of steroid hormone-dependent and autonomous tumor growth, and the transitions between the hormone-dependent and autonomous states.  相似文献   

16.
Cloned mouse mammary cell lines requiring androgens for growth in culture.   总被引:6,自引:0,他引:6  
W J Desmond  S J Wolbers  G Sato 《Cell》1976,8(1):79-86
Clonally derived cell lines have been established from the mouse mammary tumor Shionogi carcinoma 115. Physiological concentrations of androgens markedly increase the growth rate and saturation density of these clones. The cells fail to grow in 1% fetal bovine serum-supplemented medium, but addition of 10-50 nM androstanolone or testosterone results in resumption of exponential growth. Some other androgens, but not estrogens or progesterone, stimulate growth of these cells. Steroid receptors can be isolated from the cells. In addition to their effect on growth, androgens also cause a marked alteration in the cell and colony morphology of the clones.  相似文献   

17.
The effects of dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and testosterone on the growth of the androgen-dependent Shionogi SC-115 tumour in mice have been compared and the metabolites in the tumour arising from each steroid have been identified. After the transfer of SC-115 tumour cells to castrated male mice, treatment of the recipients with dihydrotestosterone produced a striking proliferative response that enabled earlier tumour detection and led to a higher tumour incidence than obtained with testosterone. At short intervals after the intravenous injection of 200muCi of [1,2-(3)H]testosterone the amounts of radioactivity in tumour, muscle and seminal vesicles were almost equal. The metabolism of [1,2-(3)H]testosterone in tumour and muscle was slight in comparison with the extensive metabolism in seminal vesciles. Whereas up to 7% of the total neutral steroid recovered from whole tumour tissue and isolated nuclei was in the form of [1,2-(3)H]dihydrotestosterone, the amount of this compound in the corresponding preparations from seminal vesciles was several times greater. When the metabolism of [1,2-(3)H]dihydrotestosterone in tumour tissue was studied, it was found that more than 60% of the total neutral steroid in both cytoplasm and nuclei consisted of [1,2-(3)H]dihydrotestosterone. Thus much higher intracellular concentrations of dihydrotestosterone occurred with the administration of this steroid than with testosterone. Tumour cell proliferation was suppressed by oestradiol and the amount of androgen in nuclei was significantly decreased by high doses of this hormone.  相似文献   

18.
Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.  相似文献   

19.
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.  相似文献   

20.
We designed and synthesized novel pyrrole-2-carboxamide derivatives as androgen antagonists. Compounds 10 and 13 bearing benzylamine or aniline at the 4-position of the pyrrole ring showed moderate androgen antagonistic activity, and inhibited the androgen-dependent growth of Shionogi carcinoma cells (SC-3). Study of the structure-activity relationships of compound 13 led to a potent androgen antagonist 36, which has higher affinity than flutamide (4) for androgen nuclear receptor (AR). Thus, pyrrole-2-carboxamide is a new scaffold for developing AR antagonists.  相似文献   

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