Correlation between hormone dependency and the regulation of epidermal growth factor receptor by tumor promoters in human mammary carcinoma cells.

The effects of the tumor promoter phorbol 12-tetradecanoate 13-acetate (TPA) on the epidermal growth factor (EGF) receptor levels were investigated in hormone-dependent (MCF-7, T-47-D, and ZR-75-1) and hormone-independent (MDA-MB-231, HBL-100, and BT-20) human mammary carcinoma cell lines. In the absence of TPA, hormone-independent cell lines contained high concentrations of low-affinity EGF receptors (apparent Kd = 8 X 10(-10) M), whereas hormone-dependent cell lines exhibited low concentrations of high-affinity receptors (apparent Kd = 1 X 10(-10) M). TPA causes a change of the receptor from a high- to the low-affinity state in hormone-dependent cell lines (MCF-7, T-47-D, and ZR-75-1), as well as in the hormone-independent HBL-100, whereas the affinity remained unchanged in MDA-MB-231 and BT-20 cells. In addition, progesterone receptor levels are decreased after TPA treatment in the hormone-dependent cell lines MCF-7, T-47-D, and ZR-75-1, whereas the estrogen receptor levels remained unchanged. Tumor promoters such as TPA or teleocidin inhibited the proliferation of these cell lines at concentrations above 10 microM with the exception of the T-47-D cells. The most sensitive cell line towards growth inhibition by tumor promoter was the hormone-dependent MCF-7 cell line. Evaluation of different TPA analogs indicated a positive correlation between the growth-inhibitory effects and their ability to stimulate the subcellular redistribution of protein kinase C activity in MCF-7 cells. These data suggest a protein kinase C-mediated down-regulation of the progesterone receptor concentration and of the EGF receptor affinity, which is supposed to mediate the mitogenic response. Furthermore, these results support the hypothesis that the tumor-derived growth factors induced by estradiol act via the EGF receptor in hormone-dependent mammary carcinoma cells.     

Current concepts concerning the role of leptin in reproductive function

Medroxyprogesterone acetate (MPA), which is frequently used as second line hormonal therapy for the treatment of metastatic breast cancer, binds with high affinity to the progesterone receptor (PR). However, the androgenic side-effects of MPA suggest that it may also activate androgen receptor (AR) regulated pathways. Treatment of the human breast cancer cell lines MDA-MB-453, ZR-75-1 and T47-D with high dose (100 nM) MPA resulted in 26-30% inhibition of cell growth, which was partially reversed by co-treatment with a 10-fold excess of the synthetic antiandrogen, anandron. Scatchard analysis demonstrated specific, high affinity (non-PR) binding of [3H]MPA to cytosols prepared from the PR-/AR+ MDA-MB-453 and PR+/AR+ ZR-75-1, but not the PR-/AR- BT-20 breast cancer cell lines. Competition of [3H]MPA binding to MDA-MB-453 cytosols by equimolar concentrations of androgens (5alpha-dihydrotestosterone (DHT), R1881) and the antiandrogen, anandron was consistent with binding of MPA to the AR. In ZR-75-1 cell cytosol fractions, DHT, R1881 and anandron only partially competed out [3H]MPA binding, suggesting that androgens displace [3H]MPA binding to AR but not to PR. Induction by MPA of AR transactivation was demonstrated in MDA-MB-453 and ZR-75-1 cells, and in the CV-1 cell line transfected with a full-length AR. In these cell lines the increased activity of the androgen responsive reporter gene (MMTV-CAT) by 1 nM MPA was fully (MDA-MB-453, CV-1) or partially (ZR-75-1) inhibited by co-culture with 1 microM anandron. These findings indicate that MPA is an AR agonist and suggest that the in vivo effects of MPA in breast cancer patients may in part be mediated by the AR.     

Cytokine (IFN-alpha, IFN-gamma, IL-1-alpha, TNF-alpha)-induced modulation of HLA cell surface expression in human breast cancer cell lines.

Cytokine-induced modulation of HLA expression on the cell surface of four human breast cancer cell lines was determined by continuous flow immunocytofluorometry with the aid of monoclonal antibodies directed to a non-polymorphic determinant of HLA class I and class II (DR) antigen. IFN-gamma and IFN-alpha were potent inducers of HLA class I in all examined cell lines, with decreasing inducibility as follows: BT-20, ZR-75-1, MCF-7 and MDA-MB-468 cells. HLA class II (DR) antigen was highly inducible by IFN-gamma in ZR-75-1 cells, followed by BT-20, MDA-MB-468 and MCF-7 cells. IFN-alpha increased the cell surface expression of DR antigen only in ZR-75-1 cells. IL-1-alpha induced a moderate level of HLA class I antigen in ZR-75-1, BT-20 and MDA-MB-468 cells, and HLA class II (DR) expression only in ZR-75-1 cells. This pattern of cell line inducibility by IL-1-alpha was similar to that induced by TNF-alpha. Differences in inducibility of HLA antigens on human breast cancer cell lines induced by different cytokines may reflect the differences in cytokine inducibility of the original tumor cells.     

Comparison of different agonists and antagonists of luteinizing hormone-releasing hormone for receptor-binding ability to rat pituitary and human breast cancer membranes

A sensitive multipoint assay capable of measuring receptors for LHRH and its analogs using 5-10 micrograms membrane protein/incubation tube was used to determine binding characteristics of different agonists and antagonists of LHRH in membranes of male rat pituitary and human breast cancer specimens. This method also permitted Scatchard analysis of the receptor binding in pellet fractions of human breast cancer biopsies remaining from estrogen and progesterone receptor assays. The potent agonist [D-Trp6]LHRH bound to at least two classes of receptor sites, one with high affinity and one with low affinity in both rat pituitary and human breast cancer samples. The analysis of displacement curves of LHRH by agonists and antagonists showed that LHRH also bound to two classes of receptor sites in pituitary and one receptor site with lower affinity in human breast cancer membranes. Among the antagonists synthesized in our laboratory, SB-030, SB-077, SB-088, and SB-090 appeared to be the most potent in displacing labeled [D-Trp6]LHRH and showed the highest binding affinity to the pituitary and breast cancer membranes. Labeled antagonists showed somewhat less affinity to membranes of pituitaries and human cancers than the agonists and bound to only a single class of receptor population. Both agonists and antagonists were able to bind to membranes of human breast cancer samples, and some antagonists were very potent in this respect. Certain LHRH agonists or antagonists could be capable of exerting direct inhibitory effects on breast cancers depending upon the presence and characteristics of LHRH receptors.     

Gonadotropin-releasing hormone receptors in human pituitary: ligand structural requirements, molecular size, and cationic effects

A specific, high affinity receptor for GnRH in human pituitaries obtained post mortem is described. The human pituitary GnRH receptor bound GnRH, a GnRH agonist [(D-Ala6,N alpha-MeLeu7,Pro9NEt)-GnRH], and a GnRH antagonist [Ac-D-Nal(2)1,D-alpha-Me-4-ClPhe2,D-3-Pal3,D-Arg6,D-Ala10 )-GnRH] with similar affinities (KdS of 4.81 nM, 0.32 nM, and 0.32 nM) to those of the rat pituitary (KdS of 4.71 nM, 0.31 nM, and 0.40 nM). A second GnRH antagonist [(D-pGlu1,D-Phe2,D-Trp3,6)-GnRH], however, was bound with a much lower affinity by the human receptor (Kd of 4.21 nM) than that of the rat (Kd of 0.09 nM). Monovalent and divalent cations affected [125I]GnRH agonist binding to rat and human pituitary receptors differently. In the presence of Mg2+ or Ca2+, binding to the human receptor was significantly lower than in the rat. At near physiological concentrations, Na+ and K+ (100 mM and 10 mM, respectively) inhibited [125I]GnRH agonist binding to the receptors to a similar extent in both species. At unphysiological concentrations (10 mM Na+ and 100 mM K+) these ions decreased binding to the human pituitary receptor to a greater extent than to the rat receptor. Using a ligand-immunoblotting technique, the human receptor or binding component of the receptor complex was found to be of greater molecular size (64,000 daltons) than that of the rat (60,000 daltons). The results show that the human and rat pituitary GnRH receptors have similar binding affinities for GnRH and certain GnRH analogs but differ in their binding of an antagonist, their sensitivity to cationic effects on GnRH agonist binding, and in the molecular size of the receptor GnRH-binding protein.     

Metabolic fate of estradiol in human mammary cancer cells in culture: estrogen sulfate formation and cooperativity exhibited by estrogen sulfotransferase

The metabolism of 17 beta-estradiol in both estrogen receptor positive and negative human breast cancer cell lines has been compared. Initial experiments in which confluent cells were exposed to 1 nM [3H]17 beta-estradiol for 24 h, revealed that the main metabolites formed by estrogen receptor positive MCF-7 and ZR-75-1 cells were 17 beta-estradiol-3-sulfate (together with lesser amounts of estrone sulfate) and estrone. In estrogen receptor negative cell lines, production of estrogen sulfates was either significantly lower (MDA-MB-231 cells) than receptor positive cells, or failed to be produced at all (MDA-MB-330 cells). In both these receptor negative cell lines, production of estrone was significantly higher than in receptor positive cells. Accumulation of estrogen sulfates resulted from attainment of a steady state between synthesis catalysed by estrogen sulfotransferase and degradation catalysed by estrogen sulfatase. The former was present in the cytosol and showed a very high affinity for 17 beta-estradiol and estrone (low nM range). Complex initial velocity versus estrogen substrate curves were obtained with enzyme purified 106-fold by affinity chromatography. Such curves were consistent with a rate equation of degree 3 or 4 and suggest the presence of cooperatively linked dependent binding sites.     

Inhibition of growth of human mammary tumor cells by potent antagonists of luteinizing hormone-releasing hormone.

Various studies support the view that analogs of luteinizing hormone-releasing hormone (LH-RH) exert some direct effects on mammary tumor cells. Recently, new LH-RH antagonists [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Hci6,D-Ala10]-LH-RH (SB-29) and [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH (SB-30), which are devoid of edematogenic effects, were synthesized. In this study, we examined whether these LH-RH antagonists inhibit the proliferation of MDA-MB-231 human mammary tumor cells in culture. [3H]Thymidine incorporation into DNA and cell number were measured. The antagonists induced up to 40% inhibition of [3H]thymidine incorporation in MDA-MB-231 cells. This inhibition was dose-dependent in the 0.3-30 microM range and could be demonstrated after 2 days of incubation in the presence of the peptides. An older antagonist, [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]-LH-RH (ORG 30276), had a lesser effect, and the agonist des-Gly10-[D-Ser(tBu)6]LH-RH ethylamide (buserelin) had no effect. The antagonists SB-29 and SB-30 also inhibited the rate of cell growth, as measured by cell number, while the LH-RH agonist buserelin had no significant effect. These results support the concept that these new LH-RH antagonists can directly inhibit the growth of human mammary tumors and thus might be suitable for the treatment of breast cancer.     

Different response of murine and human mammary tumour models to a series of diastereoisomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes

Summary A series of isomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes and cisplatin were tested on the P388 leukemia and on the murine hormone-independent MXT (M3.2) OVEX and the ovarian — hormone — dependent MXT (M3.2) mammary carcinoma for evaluating antineoplastic activity against breast cancer in vivo. Although these results were heterogeneous, a trend to the 2,6-difiuorosubstituted compound as the most active platinum complex was observed. For the development of a large-scale in vitro screening method on human breast cancer cell lines, cell number, [³H]thymidine incorporation, and crystal violet staining were evaluated as parameters for end-point determination. Chemosensitivity testing on the human breast cancer cell lines MDA-MB-231 and MCF-7 unam-biguously identified [1,2-bis(2,4-difluorophenyl)ethyle-nediamine]dichloroplatinum(II) as the complex with the highest activity in the crystal violet micro-assay. In equimolar concentration this compound was superior to cisplatin on both cell lines. The analysis of the conflicting results of this study indicates that murine mammary carcinomas are most probably unrealistic and inappropriate models for the screening of cytotoxic platinum complexes with potential activity on human breast cancer.Abbreviations PBS phosphate-buffered saline - PEG polyethlyeneglycol Dedicated to Professor Franz Lux on the occasion of his 65th birthday     

Estrogen-like activity of ginsenoside Rg1 derived from Panax notoginseng

Ginsenosides have demonstrated pharmacological effects in the central nervous, cardiovascular, and endocrine systems. We hypothesize that ginsenosides might mediate some of their actions by binding to the estrogen receptor, as they share many of the protective actions of estrogen in various physiological systems. The present study is aimed to determine whether ginsenoside Rg1 can act like an estrogen analog in stimulating human breast cancer cell growth as well as in the activation of estrogen response element-luciferase activity in HeLa cell. Rg1, but not its aglycone, stimulates [methyl-(3)H] thymidine incorporation in estrogen receptor-positive MCF-7 in a dose-dependent manner (10(-15)-10(-7) M). The stimulation of MCF-7 cell proliferation by 3 x 10(-13) M Rg1 can be blocked by 10(-6) M of the estrogen antagonist ICI 182780. Moreover, Rg1 stimulates estrogen response element-luciferase reporter gene activity in HeLa cells with an optimal dose of 3 x 10(-10) M. Such stimulation can also be blocked by 10(-6) M ICI 182780. In addition, Rg1 has no effect on [methyl-(3)H]thymidine incorporation in estrogen receptor-negative human breast cancer cells (MDA-MB-231). Furthermore, Rg1 failed to displace the specific binding of [(3)H]17 beta-estradiol to MCF-7 cell lysates, suggesting that no direct interaction of Rg1 with estrogen receptor is needed for its estrogenic action. Our results indicate that ginsenosides Rg1 has estrogen-like activity and should be classified as a novel class of potent phytoestrogen.     

Inhibition of human epithelial ovarian cancer cell growth in vitro by agonistic and antagonistic analogues of luteinizing hormone-releasing hormone.

In this study, we investigated the effects of luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6]LH-RH, LH-RH antagonist [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH (SB-75), and estradiol on the growth of human epithelial ovarian cancer cell line OV-1063. Cells were cultured under estrogen-deprived conditions. Estradiol inhibited cell proliferation, as measured by cell number at 10(-9)-10(-7) M and [3H]thymidine incorporation into DNA at 10(-13)-10(-8) M. Both LH-RH analogs inhibited cell growth dose dependently in the range 10(-8)-10(-5) M, but SB-75 induced a greater growth inhibition than [D-Trp6]LH-RH. In OV-1063 cells, 125I-labeled [D-Trp6]LH-RH was bound to one class of specific, saturable binding sites with high affinity (Kd = 1.4 +/- 0.3 nM) and low capacity (4000 binding sites per cell). 125I-labeled [D-Trp6]LH-RH could be displaced by unlabeled [D-Trp6]LH-RH and SB-75, suggesting that both analogs are bound to the same receptor on OV-1063 cells. Ligand binding was dependent on time and temperature. Receptor internalization assay showed that the ligand-receptor complex was internalized at 37 degrees C, which indicates the presence of biologically active LH-RH receptors on OV-1063 cells. These results suggest that estradiol and LH-RH analogs can suppress the growth of OV-1063 human epithelial ovarian cancer cells by a direct action and that the inhibitory effect of LH-RH analogs is mediated through the high-affinity LH-RH receptors.     

Estradiol formation from testosterone by continuously cultured human breast cancer cells.

Two human breast cancer cell lines (MCF-7 and MDA-MB-231) and one cell line derived from normal human breast (HBL-100) were examined for the presence of aromatase activity by determining the amounts of [3H]estradiol ([3H]E2) formed by cell cultures incubated with [3H]testosterone. Aromatase activity was demonstrable in both breast cancer cell lines, but estradiol synthesis was not observed in HBL-100 cultures. The [3H]E2 content of MCF-7 cultures rose as a function of incubation time and substrate concentration. Furthermore, [3H]E2 formation by this cell line was suppressed by several known inhibitors of human placental aromatase. These observations represent the first evidence that some lines of continuously cultured human breast cancer cells, like some human breast tumors, are capable of forming estrogen from an extracellular precursor steroid. Cultured breast cells may provide model systems for investigating the relative importance of intracellular estrogen formation in the regulation of human breast cancer cell growth.     

Biology of the gonadotropin-releasing hormone system in gynecological cancers.

The expression of GnRH and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary and endometrium. Dose-dependent antiproliferative effects of GnRH agonists in cell lines derived from these cancers have been observed by various investigators. GnRH antagonists also have marked antiproliferative activity in most breast, ovarian and endometrial cancer cell lines tested, indicating that the dichotomy of GnRH agonists and antagonists might not apply to the GnRH system in cancer cells. The classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of GnRH analogs in cancer cells. Rather, the GnRH receptor interacts with the mitogenic signal transduction of growth factor receptors and related oncogene products associated with tyrosine kinase activity, via activation of a phosphotyrosine phosphatase, resulting in downregulation of cancer cell proliferation. In addition, GnRH activates nuclear factor kappaB and protects the cancer cells from apoptosis. Furthermore, GnRH induces activation of the c-Jun N-terminal kinase/activator protein-1 (AP-1) pathway independent of the known AP-1 activators, protein kinase or mitogen activated protein kinase.     

Human peripheral blood mononuclear cells express gonadotropin-releasing hormone (GnRH), GnRH receptor, and interleukin-2 receptor gamma-chain messenger ribonucleic acids that are regulated by GnRH in vitro

The hypothalamic decapeptide, GnRH, plays a critical role in human reproduction. In addition to the well known effects of GnRH on pituitary cells, there is evidence supporting the presence of GnRH-binding sites in tissues other than pituitary cells, including lymphocytes. In addition, a GnRH-like substance has been found to be secreted from lymphoid cells. However, the precise nature of GnRH secretion and binding in immune cells has not been fully established. In this study, we used the RT-PCR method to examine the expression and regulation of GnRH, GnRH receptor (GnRHR), and interleukin-2 receptor gamma-chain messenger ribonucleic acids (mRNAs) in human peripheral blood mononuclear cells. It was found that human mononuclear cells expressed GnRH and GnRHR mRNAs. Nucleotide sequences of these mRNAs are identical to their hypothalamic and pituitary counterparts, respectively. In addition, GnRH and GnRHR mRNA expressions in peripheral blood mononuclear cells are regulated by GnRH and its synthetic analogs in vitro. Treatment with various concentrations of GnRH (10(-5)-10(-11) mol/L) increased GnRHR mRNA expression in a dose-dependent manner (maximal level is 158% of the untreated control value at 10(-8) mol/L GnRH; P < 0.05), but reduced GnRH mRNA levels to 69% of the untreated control value at 10(-9) mol/L GnRH (P < 0.05). Cotreatment of GnRH with a GnRH antagonist blocked these regulatory effects, indicating the receptor-mediated nature of the GnRH action. Both GnRH and GnRH agonist stimulated interleukin-2 receptor gamma-chain mRNA in a dose-dependent manner, indicating that GnRH may be involved in lymphocyte activation. In summary, these observations suggest that mRNAs encoding the pituitary form of GnRHR and the hypothalamic form of GnRH are also expressed in human peripheral blood mononuclear cells. The endogenous production of GnRH by lymphocytes may act as an autocrine or paracrine factor to regulate immune functions. Because of the presence of GnRHR on lymphocytes, exogenous GnRH analog therapy may have an impact on the immune system through these receptors.     

Characterization of GnRH receptors in bovine pituitary membranes

Bovine pituitary gonadotropin-releasing hormone (GnRH) receptors were characterized and identified utilizing a superactive GnRH analog, Buserelin [( D-Ser(t-Bu)6, des-Gly10-ethylamide]-GnRH), and a photoreactive GnRH analog, [azidobenzoyl-D- Lys6 ]-GnRH. Both analogs bind with high affinity to a single class of receptors, with apparent IC50 values of 0.5 nM and 1 nM, respectively. The binding of 125I-labeled Buserelin to pituitary membranes was inhibited, in a dose-responsive manner, by both trypsin and chymotrypsin, with the former being less effective. Neuraminidase at a concentration up to 100 micrograms/ml did not affect the binding. Lectins, such as concanavalin A and wheat-germ agglutinin, at a concentration range of 20-200 micrograms/ml had no effect on the binding, whereas soybean agglutinin at high concentrations (150 and 200 micrograms/ml) slightly inhibited the specific binding. Photoaffinity labeling of the bovine pituitary GnRH receptors resulted in the identification of two specific bands with apparent molecular weights of 60 K and 30 K daltons. The latter probably represents very low affinity binding sites. Both specific bands were sensitive to trypsin and chymotrypsin treatment but were not affected by neuraminidase treatment. These results suggest a slight difference between rat and bovine pituitary GnRH receptors.     

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.     

Characterization of gonadotropin-releasing hormone receptors in cultured rat pituitary cells

The properties of gonadotropin-releasing hormone (GnRH) receptors were analyzed in isolated pituitary cells prepared by enzymatic dispersion with trypsin or collagenase-hyaluronidase. The initial impairment of LH responses to GnRH in isolated cells prepared by both methods was reversed during culture for 2 days in multiwell vessels. However, specific binding sites for GnRH, assayed by equilibration with [125I]iodi0[D-Ser(t-BU)6]des-Gly10-GnRH N-ethylamide (GnRH-A) were demonstrable in collagenase-dispersed cells, both initially and after 2 days in culture. Cellular uptake of GnRH-A was temperature dependent, with rapid and saturable binding to a limited number of specific receptor sites with high affinity for the labeled analog (Ka = 4.0 +/- 0.8 X 10(9) M-1). These sites showed common binding specificity for GnRH-A and the native GnRH peptide, with significantly lower affinity for the natural peptide (Ka = 2.3 X 10(7) M-1). Other protein and peptide hormones, including ovine LH, ovine PRL, hCG, TRH, somatostatin, and angiotensin II (up to 10(-6) M) did not inhibit binding of GnRH-A to its receptors. Cellular binding of GnRH-A was followed by increased cGMP production and LH release within 10 min. The analog was 50 times more potent than native GnRH in stimulating LH release. The Kact values derived for GnRH and GnRH-A were 0.5 and 0.01 nM, respectively, considerably lower than the Kd values of 50 and 0.25 nM derived from receptor binding analysis. These results indicate that GnRH receptors can be identified in isolated pituitary cells, in which peptide binding is followed by increased cGMP production and LH release. The impaired LH responses in acutely dispersed pituitary cells are not due to the loss of receptor sites but to a reversible postreceptor defect. Occupancy of about 20% of the GnRH-binding sites elicits a near-maximal LH response, indicating the nonlinearity of GnRH-receptor coupling to secretory responses in the cultured gonadotroph.     

MUC1 expression in human breast cancer cells is altered by the factors affecting cell proliferation

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. In the present study, we have investigated if the factors affecting cells proliferation could influence MUC1 mucin biosynthesis and shedding from cell surface into the culture medium in two human breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ER-). Using MCF-7 line we found that estradiol at a concentration of 10(-7) M increased [3H]glucosamine incorporation into mucin in cell lysate approximately twofold in comparison with control cultures, and a similar increase was observed in the culture medium. The selective estrogen receptor modulator, tamoxifen (at concentrations of 10(-6) M and 10(-5) M) had a little inhibitory effect. MDA-MB-231 cells in culture were stimulated with phorbol ester PMA, the protein kinase C activator. We noted that PMA greatly stimulated MUC1 synthesis and its shedding to culture medium and that this effect was abolished by protein kinase C specific inhibitor--bisindolylmaleimide.