19 research outputs found

    Effects of antiandrogens on transformation and transcription activation of wild-type and mutated (LNCaP) androgen receptors

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    LNCaP cells contain androgen receptors with a mutation in the steroid binding domain (Thr 868 changed to Ala) resulting in a changed hormone specificity. Both the wild-type and mutated androgen receptors were transfected into COS cells. Transcription activation was studied in cells co-transfected with an androgen sensitive reporter (CAT) gene. The wild-type androgen receptor was activated by the agonist R1881, but the antiandrogens did not enhance transcription apart from a partial agonistic effect at high concentrations of cyproterone acetate. The mutated androgen receptor was fully activated by R1881, cypoterone acetate and hydroxyflutamide, but not by ICI 176,334. Receptor transformation to a tight nuclear binding state was studied by preparation of detergent washed nuclei and Western blotting with a specific antibody against the androgen receptor. Nuclei of COS cells transfected with wild-type receptor retained the receptor when the cells had been treated with the agonist R1881, partially retained receptors when treated with antiandrogen cyproterone acetate, but did not retain receptor when treated with hydroxyflutamide or ICI 176,334. The cells transfected with the mutated receptor additionally retained nuclear receptors after treatment with hydroxyflutamide. We conclude that each one of the three antiandrogens tested displayed different characteristics with respect to its effect on transformation and transcription activation

    Stimulatory effects of antiandrogens on LNCaP human prostate tumor cell growth, EGF-receptor level and acid phosphatase secretion

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    Abstract LNCaP cells (derived from a lymph node carcinoma of the human prostate) show androgen responsive growth. Progestagens, estradiol and antiandrogens competed with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen-sensitive systems. Optimal growth (3–4 fold increase in DNA content of 6 day cell cultures vs controls) was observed after addition of the synthetic androgen R1881 (0.1 nM). Both steroidal and non-steroidal antiandrogens did not suppress the androgen responsive growth. At a concentration of 10 nM cyproterone acetate or 100 nM RU 23908, growth was even stimulated to an extent comparable to that observed after addition of androgen. Cyproterone acetate and RU 23908 also increased the number of epidermal growth factor receptors expressed at the cell surface to a comparable level as did the androgen. Like androgens, cyproterone acetate, RU 23908 or estradiol stimulated the secretion per cell of prostate specific acid phosphatase in the culture fluid. In conclusion, antiandrogens can exert striking stimulatory effects on the proliferation of LNCaP cells probably due to a defective androgen receptor system. It is discussed that comparable changes in the specificity of the androgen receptor in prostate cancer cells may give these cells an advantage in growth rate and may contribute to development of tumors characterized as hormone independent

    特許ライセンスをめぐる最近の立法動向

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    markdownabstract__Abstract__ The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-α/ml or 20 ng basic FGF/ml. TGF-β (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-α-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells in therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-β did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGFα (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGFga-likely activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mut of the androgen receptor

    Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: High affinity for progestagenic and estrogenic steroids

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    Abstract LNCaP tumor cells, derived from a metastatic lesion of a human prostatic carcinoma, are androgen-sensitive in cell culture. Although increase in growth rate is observed with low doses of progestagens or estradiol, these cells contain exclusively androgen receptors. In the present study the binding affinity of different ligands for both non-DNA- and DNA-binding (transformed) forms of the androgen receptor were analyzed. The cytosolic (non-transformed) form of the receptor displayed an abnormal high affinity for progestagens and estradiol when compared with the cytosolic androgen receptor from other sources. Subsequently the non-transformed forms of the androgen receptor obtained from LNCaP cell nuclei was studied. A high binding affinity was found not only for dihydrotestosterone, but also for progesterone and the synthetic progestagen R5020 (relative binding affinity 42% and 10% of dihydrotestosterone). The binding characteristics of the transformed androgen receptor were examined in intact cells at 37°C. LNCaP cells were compared in this respect with COS cells containing the cloned human androgen receptor, normal human skin fibroblasts and PC3 (prostate) and NHIK (cervix) human tumor cell lines. The affinity of the transformed androgen receptors for the progestagen R5020 in LNCaP cells was significantly higher than in the other cell systems, although the differences were less pronounced than for the non-transformed receptor form. In conclusion: the LNCaP tumor cells contain an androgen receptor with an abnormal binding site. This might be due to a mutation and/or a post-transcriptional effect

    Breast cancer oestrogen independence mediated by BCAR1 or BCAR3 genes is transmitted through mechanisms distinct from the oestrogen receptor signalling pathway or the epidermal growth factor receptor signalling pathway

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    INTRODUCTION: Tamoxifen is effective for endocrine treatment of oestrogen receptor-positive breast cancers but ultimately fails due to the development of resistance. A functional screen in human breast cancer cells identified two BCAR genes causing oestrogen-independent proliferation. The BCAR1 and BCAR3 genes both encode components of intracellular signal transduction, but their direct effect on breast cancer cell proliferation is not known. The aim of this study was to investigate the growth control mediated by these BCAR genes by gene expression profiling. METHODS: We have measured the expression changes induced by overexpression of the BCAR1 or BCAR3 gene in ZR-75-1 cells and have made direct comparisons with the expression changes after cell stimulation with oestrogen or epidermal growth factor (EGF). A comparison with published gene expression data of cell models and breast tumours is made. RESULTS: Relatively few changes in gene expression were detected in the BCAR-transfected cells, in comparison with the extensive and distinct differences in gene expression induced by oestrogen or EGF. Both BCAR1 and BCAR3 regulate discrete sets of genes in these ZR-75-1-derived cells, indicating that the proliferation signalling proceeds along distinct pathways. Oestrogen-regulated genes in our cell model showed general concordance with reported data of cell models and gene expression association with oestrogen receptor status of breast tumours. CONCLUSIONS: The direct comparison of the expression profiles of BCAR transfectants and oestrogen or EGF-stimulated cells strongly suggests that anti-oestrogen-resistant cell proliferation is not caused by alternative activation of the oestrogen receptor or by the epidermal growth factor receptor signalling pathway

    Mechanisms of action of androgen receptor agonists and antagonists

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    The aim of the studies described in the next chapters was, first, to prove that the AR in LNCaP cells is abnormal with respect to ligand binding characteristics, and to find an explanation for this defect. Second, it was investigated whether this aberration could account for the growth stimulating effects of antiandrogens on this cell line. Third, the effects of both androgens and antiandrogens were investigated at the biochemical level, with much emphasis on receptor interactions with other (heat-shock) proteins. Chapter 2 describes investigations to study the binding affmities of several steroidal and non-steroidal ligands for the AR in LNCaP cells. These binding affinities were compared with the binding affmities for the AR from other sources, including cells expressing wild type AR (Chang eta!., 1988; Lubalm eta!., 1988; Trapman eta!., 1988; Faber et a!., 1989). From studies with nuclear preparations, devoid of cytoplasmic contantinations, it was concluded that the binding affinity of the AR in LNCaP cells was abnormal. The third chapter describes that the AR gene in LNCaP cells contains a mutation. The expression ofthe mutant receptor in LNCaP cells was confirmed by eDNA sequence analysis. In transfection studies, the binding specificity of the mutant receptor was compared with the binding specificity of the wild type receptor expressed in the same cell type. Also the ability of both the mutant and wild type receptor to activate transcription from an AR responsive construct in response to androgens, antiandrogens, progestins and estrogens was investigated. One antiandrogen, ICI 176 334 ("casodex", a trade mark of!CI Pharmaceuticals), was found which could not stimulate growth of LNCaP cells, but inhibited the effect mediated by androgens (Chapter 4). It was investigated whether there is a difference between antiandrogens such as hydroxyflutamide, which induce growth of LNCaP cells, other hand. The ability of these compounds to provoke a dissociation of the AR-heatshock protein-complex was studied. In addition, it was investigated whether the three heat-shock proteins hsp90, hsp70, and hsp56 could be detected in the heteromeric complexes. In Chapter 5 of this thesis, the effects of incubation of LNCaP cells with androgens on the AR-heat-shock protein-complex is described. Both changes in complex-size and composition, and changes in affinity of the receptor for the nucleus were analyzed. In addition, the development of an antibody against part of the DNA-binding domain of the AR is described. This antibody was used to examine whether its epitope was exposed on the surface of untransformed and transformed ARs. It was also tested whether this antibody could be used to specifically precipitate wholly or partially transformed receptors. Finally, in Chapter 6, the results from the former chapters are discussed in a broader context. The effects of the mutation in the AR of LNCaP cells on results obtained with estrogens, progestins and antiandrogens are discussed. The possible role of the different heat-shock proteins in receptor transformation is considered. Suggestions are made for future investigation

    Studies on the human prostatic cancer cell line LNCaP

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    The effects of androgens, antiandrogens, and other steroid hormones on growth of the human prostate cancer cell line LNCaP were studied. Despite the absence of receptors for progesterone and estradiol, the growth rate of the androgen responsive LNCaP-FGC cells increased when cultured in the presence of either estrogens or progestagens. In addition, most antiandrogens were also growth stimulators. This aberrant response was due to a threonine to alanine substitution at amino acid position 868 in the steroid binding domain of the androgen receptor (AR). Only the antiandrogen ICI 176 334 could block transcription and cell growth by the mutant receptor. By immunoprecipitation of the AR from LNCaP cells with the specific antibody F39.4.1 and Western blotting, three types of heat-shock proteins co-precipitated: hsp90, hsp70 and hsp56. This co-isolation could be prevented by pre-incubating the cells with androgens or with the antiandrogen hydroxyflutamide. Only the antiandrogen ICI 176 334 could block the effect of androgens on complex dissociation and prevent tight nuclear binding of the AR. Hydroxyflutamide could only inhibit tight nuclear binding of the wild-type AR. Therefore, in LNCaP cells the mutation in the steroid binding domain of the AR prevents a blockade of receptor function by most antiandrogens, but not by ICI 176 334, probably because of a different mechanism by which this compound blocks receptor function
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