The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens

The human prostate tumor cell line LNCaP containd an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor proteon and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induced reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

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-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-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 is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (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 TGF alpha-like 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 mutated amino acid in the steroid binding domain of the androgen receptor.     

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

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.     

雄激素受体信号转导途径及其在前列腺癌发展中的作用

前列腺癌是西方男性发病率最高的癌症之一,在采用雄激素阻断疗法后,大部分患者的病情可得到控制,但经过一段时间又会转变为雄激素非依赖型前列腺癌。雄激素受体(AR)在前列腺细胞中扮演重要的角色,它可调节大量基因的表达。在前列腺癌由雄激素依赖型向雄激素非依赖型的转变过程中,AR及其信号途径通过多种方式发挥作用,AR基因的扩增、AR的突变,以及与共激活子之间作用的改变都可能使细胞获得雄激素非依赖型的生长能力。此外,AR还受到多肽生长因子和细胞因子等的调节,表现激素非依赖型的转录激活活性。AR在前列腺癌中作用的阐明对前列腺癌的诊断与治疗有着重大的意义。     

Effects of the Antiandrogen Flutamide on the Expression of Protein Kinase C Isoenzymes in LNCaP and PC3 Human Prostate Cancer Cells

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (alpha, beta1, epsilon, zeta) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 microM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of alpha, beta1 and zeta, but not epsilon, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCbeta1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.     

A differential ligand-mediated response of green fluorescent protein-tagged androgen receptor in living prostate cancer and non-prostate cancer cell lines.

Androgen has been shown to promote the proliferation of prostate cancer through the action of the androgen receptor (AR). Mutation (T877A) of the AR gene found in an androgen-sensitive prostate cancer cell line, LNCaP, has been postulated to be involved in hypersensitivity and loss of specificity for androgen. In the present study, trafficking of AR and AR (T877A) in living prostate and non-prostate cancer cell lines under high and low concentrations of androgen and antiandrogen was investigated by tagging green fluorescent protein (GFP) to the receptors. In the presence of a high concentration of androgen, AR-GFP localized in the nucleus by forming discrete clusters in all cell lines. AR (T877A)-GFP was also translocated to the nucleus in LNCaP and COS-1 cells by the addition of a high concentration of androgen. In contrast, in the presence of a low concentration of androgen, the translocation of AR-GFP and AR (T877A)-GFP was observed in LNCaP cells, but not in COS-1 cells. Upon the addition of antiandrogen, AR-GFP was translocated to the nucleus but did not form subnuclear foci in both COS-1 and LNCaP cells, whereas AR (T877A)-GFP in both cells was translocated to the nucleus with subnuclear foci. The present study demonstrates the differential response of nuclear trafficking of AR and its mutant in prostate cancer cell lines and COS cells, and the subcellular and subnuclear compartmentalization provide important information on the sensitivity of the AR mutation.     

C19-steroids as androgen receptor modulators: design, discovery, and structure-activity relationship of new steroidal androgen receptor antagonists

Dehydroepiandrosterone (DHEA), the most abundant steroid in human circulating blood, is metabolized to sex hormones and other C19-steroids. Our previous collaborative study demonstrated that androst-5-ene-3beta,17beta-diol (Adiol) and androst-4-ene-3,17-dione (Adione), metabolites of DHEA, can activate androgen receptor (AR) target genes. Adiol is maintained at a high concentration in prostate cancer tissue; even after androgen deprivation therapy and its androgen activity is not inhibited by the antiandrogens currently used to treat prostate cancer patients. We have synthesized possible metabolites of DHEA and several synthetic analogues and evaluated their role in androgen receptor transactivation to identify AR modulators. Steroids with low androgenic potential in PC-3 cell lines were evaluated for anti-dihydrotestosterone (DHT) and anti-Adiol activity. We discovered three potent antiandrogens: 3beta-acetoxyandrosta-1,5-diene-17-one 17-ethylene ketal (ADEK), androsta-1,4-diene-3,17-dione 17-ethylene ketal (OAK), and 3beta-hydroxyandrosta-5,16-diene (HAD) that antagonized the effects of DHT as well as of Adiol on the growth of LNCaP cells and on the expression of prostate-specific antigen (PSA). In vivo tests of these compounds will reveal their potential as potent antiandrogens for the treatment of prostate cancer.     

Hormone-dependent androgen receptor phosphorylation is accompanied by receptor transformation in human lymph node carcinoma of the prostate cells

Phosphorylation of the androgen receptor was investigated in the absence of hormone as well as during and after transformation of the receptor to the tight nuclear binding form. Human prostate tumor cells (LNCaP) were labeled for 4 h with [32P]orthophosphate in the presence or absence of steroid. Subsequently, androgen receptors were immunoprecipitated either from total cell lysates or from nuclear extracts using a specific monoclonal antibody. The immunoprecipitated receptor preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, using a polyclonal antiserum, and autoradiography. It was observed that the androgen receptor is already phosphorylated in the absence of hormone, but undergoes a hormone-induced additional phosphorylation. After administration of 10 nM R1881, a 1.8-fold increase in phosphorylation over nonstimulated control cells was reached. Moreover, the amount of nuclear extractable androgen receptor was increased; the acquisition of tight nuclear binding capacity was accompanied by hormone-induced receptor phosphorylation.