Phenol Red-free Medium Research Articles

Estradiol regulates monocyte chemotactic protein-1 in human coronary artery smooth muscle cells: a mechanism for its antiatherogenic effect.

The protective effect of estrogen against early atherosclerosis in animal models is well documented, but the mechanisms responsible for this effect are not well understood. The earliest recognizable event in the pathogenesis of atherosclerosis is an increased recruitment of macrophages into the arterial subendothelium. Macrophages first play a protective role by removing low-density lipoproteins, but when the cholesterol is in excess, macrophages are converted into foam cells and form atheromas. Recent human and animal data indicate that the recruitment of macrophages to the arterial wall is mediated by monocyte chemotactic protein-1 (MCP-1). We hypothesized that one of the mechanisms of estrogen's protective effect against atherosclerosis may be the down-regulation of MCP-1 expression in the arterial wall. Human coronary artery smooth muscle cells were replicated to confluence in smooth muscle cell basal medium supplemented with growth factors and 5% fetal bovine serum. Before each experiment, cells were incubated for 24 h with phenol red-free medium containing 5% charcoal-stripped calf serum, and then they were treated with various concentrations of 17beta-estradiol as well as selective estrogen receptor (ER) modulators, raloxifene and tamoxifen. MCP-1 messenger ribonucleic acid (mRNA) levels were quantified by Northern blots. MCP-1 protein was quantified using an enzyme-linked immunosorbent assay. ER expression was evaluated by reverse transcriptase-polymerase chain reaction. Human coronary artery smooth muscle cells expressed MCP-1 mRNA and produced MCP-1 protein. Estradiol induced up to 40% inhibition in mRNA expression at concentrations 10-9 M and higher. Raloxifene and tamoxifen also resulted in an inhibition, but the inhibition was less than when induced by estradiol. Estradiol also inhibited the MCP-1 protein production in a concentration-dependent manner (p < 0.05). Coronary smooth muscle cells expressed both ERalpha and ERbeta. Our findings suggest that one of the mechanisms by which estrogen prevents atherosclerosis is by down-regulating MCP-1 expression, thus decreasing macrophage recruitment to the arterial wall.

Estradiol 17β inhibition of LDL oxidation and endothelial cell cytotoxicity is opposed by progestins to different degrees

Progestins oppose the effects of estrogens in many biological systems, but it is not known if progestins oppose the antioxidant effects of estrogen and to differing degrees. To test these questions, the effects of various sex steroids on LDL oxidation and cytotoxicity were studied in the absence or presence of endothelial cells. Freshly isolated LDL was incubated in the presence of Cu ++ in the absence or presence of cultured bovine aortic endothelial cells in phenol red-free medium and without or with hormones in 0.5% ethanol. The hormones included 17β-estradiol (E 2), progesterone (Pg), norgestimate (NGM), levonorgestrel (LNG), and medroxyprogesterone acetate (MPA). LDL oxidation was measured as formation of conjugated dienes, lipid peroxides, and TBARS, and cyotoxicity by tetrazolium salt reduction (MTT reduction). Progestins diminished conjugated diene lag phase, accelerated lipid peroxide and TBARS production in the absence and presence of cells and accelerated cytotoxicity. When E 2 and progestin were incubated with cells at a molar ratio of 1:5, lipid peroxides were reduced from baseline by E 2 alone 31%, E 2/Pg 29%, E 2/NGM 16%, E 2/LNG 9% (all P<0.05 or more) and E 2/MPA 8% (ns) (E 2 or E 2/Pg>E 2/NGM, E 2/LNG and E 2/MPA [ P<0.001]; E 2/NGM>E 2/LNG or E 2/MPA [ P<0.05]). MTT reduction followed a similar gradient, greatest with E 2 alone, least with E 2/MPA. Conclusions: Progestins promote LDL oxidation and, conjointly, endothelial cell cytotoxicity. Progestins oppose the antioxidant and cytoprotective effects of estrogen when given in combination. MPA and LNG have the strongest prooxidant and cytotoxic effects, which may limit the cardiovascular benefit of estrogen during combined administration in vivo.

Androgens modulate interleukin-6 production by gingival fibroblasts in vitro.

Pregnancy and puberty gingivitis have been attributed to increased concentrations of circulating sex hormones. This inflammatory gingival condition is accompanied by the local production of cytokines. The aims of this in vitro study were to assess, in the presence or absence of testosterone (T) or dihydrotestosterone (DHT), the production of interleukin-6 (IL-6) by human gingival fibroblasts (hGF), and to evaluate the effects of flutamide (a common anti-androgen) in this system. The effects of the androgens, T and DHT, on IL-6 production were measured in vitro in serum-free, phenol red-free medium. Cells were incubated with or without androgens for 72 hours; the concentration of IL-6 secreted into the medium after an additional 24-hour challenge with IL-1beta plus hormones was estimated by radioimmunoassay. The reverse transcription polymerase chain reaction was used to examine hGF and periodontal ligament cells (PDL) for the presence of androgen receptor. In serum-free medium, T and DHT at concentrations of 5 x 10(-8) to 10(-7)M significantly (P <0.05) inhibited IL-6 production by hGF. Flutamide, up to concentrations of 2 x 10(-5)M, did not reverse this inhibition. The androgen receptor was identified in both hGF and PDL. We concluded that elevated levels of androgens, specifically testosterone and dihydrotestosterone, could affect the stromal cell response to an inflammatory challenge by downregulation of IL-6 production. This in vitro study lends support to the hypothesis that increased hormones during pregnancy or puberty could modulate the development of localized inflammation.

Optimization of Treatment Conditions for Studying the Anticancer Effects of Retinoids Using Pancreatic Adenocarcinoma as a Model

Retinoids are natural differentiation-inducing compounds that are promising as anticancer agents. Cancer cell lines are valuable in the investigation of the potential of retinoids for the treatment of specific cancers. However, using different treatment conditions but the same cell lines, investigators have produced markedly contradictory results for the effectiveness of retinoids. The present study examined different factors in the treatment conditions that may have masked or interfered with the effects of retinoids and, thereby, resulted in this conflict. Our studies revealed that the effects of retinoids on cancer cell proliferation were influenced by serum, the choice of vehicle (DMSO vs ethanol) and its concentration, phenol red, the degree of cellular confluence, and the method of assessing proliferation (cell number or [3H]thymidine uptake vs the MTT assay). Optimized conditions were the use of serum-free, ethanol-free, and phenol red-free media, investigating cells in the log phase of growth, using ≤0.01% DMSO as the vehicle, and monitoring proliferation by cell number or [3H]thymidine incorporation into DNA measured after TCA precipitation. Using these conditions, retinoids were found to exhibit potent antiproliferative effects in pancreatic cancer cells with a variety of degrees of differentiation, even in cell lines previously documented as being retinoid resistant. Retinoids also induced morphological changes and cellular death that may indicate terminal differentiation and apoptosis.

Growth and phenotypic characterization of porcine coronary artery smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation significantly contributes to atherosclerotic plaque formation and limits the success rate of percutaneous transluminal coronary angioplasty. We derived a population of porcine coronary artery SMCs to characterize VSMC proliferation and phenotype in preparation to study the molecular actions of VSMC mitogens and antiproliferative agents. Growth assays were designed to minimize the estrogen content in the culture medium, since this steroid hormone significantly influences VSMC growth and the expression of VSMC mitogens and their receptors. Culture conditions were identified such that this criterion was achieved while maintaining a significant VSMC growth rate. Cells cultured in serum-free medium, regardless of growth factor supplements, did not remain adherent to a plastic culture substrate, nor did they proliferate. Dextran-coated charcoal (DCC)-treated sera, including fetal bovine, calf, and porcine, supported VSMC adhesion, but not growth. Whole fetal bovine serum (FBS) produced the best proliferative response. A type-I collagen-coated culture surface significantly enhanced VSMC growth, but only in culture medium containing non-DCC-treated FBS. Flow cytometry analyses confirmed the mitogenic effects of this substrate. The VSMCs exhibited a morphological change on type-I collagen, but this was not accompanied by a change in VSMC phenotype. Our data indicate that culture of these porcine coronary artery SMCs in 2.5% FBS plus 10 ng platelet-derived growth factor-BB per ml in phenol red-free medium on type-I collagen may be the optimal conditions for studying the molecular aspects of VSMC mitogens and antiproliferative agents.

Negative regulation of N-cadherin-mediated cell-cell adhesion by the estrogen receptor signaling pathway in rat pituitary GH3 cells.

The ability of the estrogen receptor signaling pathway to regulate cell-cell adhesion, and N-cadherin and beta-catenin expression was examined in rat somatolactotropic GH3 cells cultured in serum-free, phenol red-free medium (SFM). Estradiol-17beta (E2) promoted a nonadherent phenotype, whereas the steroidal antiestrogen, ICI 182,780, induced the formation of tightly adherent aggregates of cells. The antiestrogen-induced cell-cell adhesion was associated with the presence of adherens junctions, and was Ca2+-dependent. E2 reduced surface N-cadherin protein to barely detectable levels, whereas ICI 182,780-treated cells displayed abundant punctate immunoreactive N-cadherin. Antiestrogen failed to induce adhesion in the presence of a blocking antibody to N-cadherin. ICI 182,780 increased the protein levels for N-cadherin and the cadherin-binding protein, beta-catenin, by twofold over SFM controls or E2-treated samples. ICI 182,780 also increased the mRNA levels for N-cadherin and beta-catenin by two- to fivefold. In GH3 cells cultured in growth medium, ICI 182,780 increased N-cadherin and beta-catenin levels by twofold over untreated controls, and inhibited cell proliferation by 53%. These results provide the first demonstration of the regulation of N-cadherin-mediated cell-cell adhesion by the estrogen receptor (ER) signaling pathway in pituitary somatolactotrophs through the coordinate regulation of N-cadherin and beta-catenin expression. The inverse relationship between ICI 182,780-induced adhesion and proliferation raises the possibility that these two processes are functionally related.

Response of primary rabbit kidney proximal tubule cells to estrogens.

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red-free medium. 17beta-estradiol was observed to stimulate growth at dosages as low as 10(-10) M. The growth stimulatory effects of 17beta-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10(-9) to 10(-8) M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17beta-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17beta-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17beta-estradiol. 17beta-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures.

Effects of Tamoxifen, Toremifene and Chloroquine on the Lysosomal Enzymes in Cultured Retinal Pigment Epithelial Cells

Retinal pigment epithelial cells carry out phagocytosis and digestion of material shed from the photoreceptor outer segments. In this process, the integrity of lysosomal enzymes is of major importance. In the present study the effects of tamoxifen, toremifene and chloroquine on the activity of two lysosomal enzymes (cathepsin D and N-acetyl-beta-D-glucosaminidase) in the retinal pigment epithelial cells were studied. Retinal pigment epithelial cells from pig eyes were cultured for two weeks in Dulbecco's Modified Eagle Medium, after which the cells were exposed to 1-40 microM concentrations of tamoxifen citrate, toremifene citrate and chloroquine diphosphate. To eliminate possible medium-borne oestrogenic mechanisms, the test was repeated using phenol red-free medium with charcoal-stripped fetal calf serum. The exposure time was one week, after which the lysosomal enzymes cathepsin D and N-acetyl-beta-glucosaminidase were determined. Cellular injuries were assessed by quantifying the leakage of lactate dehydrogenase into the culture medium. Cathepsin D and N-acetyl-beta-D-glucosaminidase showed different sensitivities to tamoxifen, toremifene and chloroquine. The main lysosomal protease cathepsin D was more sensitive than N-acetyl-beta-D-glucosaminidase to the effects of tamoxifen and toremifene, possibly due to their antioestrogenic properties. The phenol red-free medium with charcoal-stripped serum seemed to make the drugs more effective than the reference medium. Chloroquine had only a minor effect on the lysosomal protease cathepsin D, but a clearer effect could be seen on N-acetyl-beta-glucosaminidase.

Hormonal modulation of Ishikawa cells during three-dimensional growth in vitro.

The Ishikawa endometrial cancer cell line is hormonally responsive, expressing estrogen and progesterone receptors (ER, PR) when grown in traditional monolayer culture. The purpose of this paper is to demonstrate a three-dimensional spheroid culture system for cancer cells. We used this system to determine the response of the Ishikawa cell line to estradiol-17 beta (E), tamoxifen (T), megestrol acetate (MA), and progesterone (P). Ishikawa cells were incubated in polyurethane culture bags using phenol red-free media containing ethanol (0.1%, controls), E (1 mumol, or 1 nmol), T (1 mumol, or 10 nmol), MA (1 mumol, or 10 nmol), or P (1 mumol). Cellular morphology was assessed by hematoxylin and eosin staining, and expression of estrogen and progesterone receptors was determined immunohistochemically using an immunoperoxidase technique. Cells in control cultures demonstrated minimal organization and lacked hormone receptors. In contrast, cells exposed to either E or T displayed significant glandular formation, with multicellular, microvilli-rich, columnar epithelia exhibiting polarized nuclear arrangements. Within 4 weeks, E- and T-treated cultures showed upregulated nuclear staining for PR, with little ER present. Cells treated with MA or P showed less glandular organization but expressed ER with PR downregulation. These data support the use of this novel three-dimensional culture system to study the modulation of tumor cell biologic activity in response to hormonal agents. Future applications of this model include examining in vitro responsiveness of cancer cell lines to additional biologic agents and chemotherapeutic regimens.

In Vitro Secretion of Cytokines by Human Bone Marrow: Effects of Age and Estrogen Status1

It has been proposed that cytokines mediate the acceleration of bone loss following menopause. Because of the intimate relationship between bone marrow stromal cells and bone tissue, it is possible that marrow cells and their products contribute to the bone microenvironment and influence the regulation of bone cell differentiation and activity. We examined the production of cytokines by bone marrow stromal cells from a total of 37 women and 15 men undergoing total hip replacement for noninflammatory joint disease. Low-density mononuclear cells were isolated from bone marrow and were cultured in phenol red-free alpha MEM medium supplemented with 10% FBS and antibiotics. Constitutive secretion of interleukin-6 (IL-6) was positively correlated with age in a series of 8 women and 5 men measured by bioassay (r = 0.98; P < 0.01) and in a series of 18 women and 10 men measured by immunoassay (r = 0.56; P < 0.01). The pattern of cytokine production by bone marrow stromal cells was examined in detail in 23 postmenopausal women, aged 49-88 yr. Basal secretion of immunoreactive IL-6 and IL-11, but not granulocyte-macrophage colony-stimulating factor, increased with time in culture. Exogenous IL-1 beta stimulated secretion of IL-6 and IL-11 in a saturable, dose-dependent manner. Secretion of soluble IL-6 receptor was not correlated with secretion of IL-6, either constitutively or in the presence of IL-1 beta. In 4 of 14 samples, IL-1 beta also stimulated secretion of granulocyte-macrophage colony-stimulating factor. IL-1 beta was undetectable in 7 of 9 cultures during the 2-week culture period. IL-6 did not stimulate secretion of IL-1 beta in the 7 cultures tested. Cells were dependent upon serum for viability and growth and were not sustained by a serum substitute (1% insulin-transferrin-selenium-BSA). Cells grown in medium with 10% FBS and supplemented with 1% insulin-transferrin-selenium-BSA secreted 10-fold more IL-6 than cells grown in serum alone. Marrow from 7 women receiving estrogen replacement therapy showed lower constitutive secretion of IL-6 (75%; P < 0.006) and IL-11 (43%; P < 0.05) than marrow from age-matched controls and had blunted stimulation of IL-6 and IL-11 secretion by exogenous IL-1 beta. These data indicate distinct patterns of cytokine production by human marrow stromal cultures dependent upon age and estrogen status.

Establishment of a mouse Sertoli cell line producing rat ABP : A. Ducray, M. Bloquel, † K. Hess, ‡ G. L. Hammond, § H. Gerard,∗ and A. Gerard.∗ ∗Faculté de Médecine-Nancy I, France, Histologie-Embryologie; †IUT BA Santé; ‡Biochimie; and §London Cancer Research, Ontario, Canada

Using a bone organ culture system that shows mineralization in vitro, we investigated whether 17β-estradiol dose-dependently increases calcium content in cultured calvarial bones in serum-free medium. Fetal mouse parietal bones (3 × 3 mm) were cultured in phenol red-free BGJ medium containing phosphate (3–4 μmol/L), calcium (1–1.25 mmol/L) insulin (6μg/mL), and transferrin (6μg/mL) for 4–5 days. Under these culture conditions, the calcium content of the cultured bones (at dissection 34.0 t 4.6 μg/bone [mean ± SD], n = 50) increased by 15–20 μg during 4–5 days of culture. 17β-Estradiol increased the calcium content significantly at 10−12 to 10−11 mol/L, but not at lower (10−13 mol/L) or higher (10−10 to 10−9 mol/L) concentrations. 17α-Estradiol had no effect. The stimulatory effect of 17β-estradiol was completely inhibited by the antiestrogen agent ICI-182,780. The anabolic effect of 17β-estradiol was elicited not only in bones from females but also in those from males. 17β-Estradiol had no significant effect on 45Ca release from prelabeled parietal bones. Furthermore, light- and electron-microscopic examinations revealed that bone mineralization proceeded through formation of matrix vesicles, without any metastatic or dystrophic calcification. These in vitro findings suggest that 17β-estradiol elicits small, but reproducible, direct effects on calcium content in the parietal bones not only in female but also in male fetal mice at physiological-free E2 concentrations (10−12−10−11 mol/L), which is attainable in serum of normal human subjects. In contrast to in vivo studies, pharmacological doses of 17β-estradiol had no anabolic effect on parietal bones. The mechanism of such a biphasic effect of estrogens remains to be elucidated.

Rapid and irreversible loss of estrogen receptor in human osteoblast-like cells following culture in phenol red-free medium

Rapid and irreversible loss of estrogen receptor in human osteoblast-like cells following culture in phenol red-free medium

Estrogen regulates somatostatin receptor subtype 2 messenger ribonucleic acid expression in human breast cancer cells.

Somatostatin (SRIF) and its analogs exhibit antiproliferative effects that are mediated by SRIF receptors (sst) present in responsive normal and neoplastic tissue including breast cancer. However, information regarding regulation of sst gene expression in cancer cells and modulation of SRIF binding is limited. In the present study we have determined the pattern of sst subtype messenger RNA (mRNA) expression in human breast cancer cells. Furthermore, we investigated the effect of 17beta-Estradiol (E2) treatment on steady state levels of sst mRNA by solution hybridization/nuclease protection analysis and on SRIF binding to membranes of treated cells by receptor binding assay. sst2 mRNA was highly expressed in T47D, ZR75-1, and MDA MB231 cells. Transcripts for sst1 were also detected at very low levels in ZR75-1 cells, whereas sst5 mRNA was expressed at low levels in MCF-7 cells. No sst subtype was detected in MDA MB 435s cells. When the estrogen receptor (ER)(+) cell lines T47D and ZR75-1 were cultured in phenol red-free media plus CS-FCS, sst2 mRNA levels decreased by 60-80% compared with complete serum controls. Adding E2 restored sst2 mRNA levels to control in both cell lines. Moreover, the effect of E2 on sst2 gene expression in T47D and ZR75-1 cells was dose- and time-dependent. In contrast, neither culturing in phenol red-free media plus CS-FCS nor E2 influenced sst2 expression in the ER(-) cell line MDA MB231. E2-induced regulation of SRIF binding and sst2 mRNA expression occurred in a parallel manner in T47D cells but were dissociated in ZR75-1 cells. The pure antiestrogen ICI 182 780 inhibited E2-induced sst2 expression in both cell lines. The antiestrogen 4OH tamoxifen showed strong estrogen-like effects on sst2 mRNA expression in T47D cells, while acting as a potent antiestrogen in ZR75-1 cells. Thus, these data suggest that E2 regulates sst2 expression in human breast cancer cell lines through the ER. The human breast cancer cell lines provide a useful model to examine the molecular mechanisms involved in E2 regulation of sst2 expression.

17 beta-estradiol increases calcium content in fetal mouse parietal bones cultured in serum-free medium only at physiological concentrations.

Using a bone organ culture system that shows mineralization in vitro, we investigated whether 17 beta-estradiol dose-dependently increases calcium content in cultured calvarial bones in serum-free medium. Fetal mouse parietal bones (3 x 3 mm) were cultured in phenol red-free BGJ medium containing phosphate (3-4 mmol/L), calcium (1-1.25 mmol/L), insulin (6 micrograms/ML), and transferrin (6 micrograms/mL) for 4-5 days. Under these culture conditions, the calcium content of the cultured bones (at dissection 34.0 +/- 4.6 micrograms/bone [mean +/- SD], n = 50) increased by 15-20 micrograms during 4-5 days of culture. 17 beta-Estradiol increased the calcium content significantly at 10(-12) to 10(-11) mol/L, but not at lower (10(113) mol/L) or higher (10(-10) to 10(-9) mol/L) concentrations. 17 alpha-Estradiol had no effect. The stimulatory effect of 17 beta-estradiol was completely inhibited by the antiestrogen agent ICI-182,780. The anabolic effect of 17 beta-estradiol was elicited not only in bones from females but also in those from males. 17 beta-Estradiol had no significant effect on 45Ca release from prelabeled parietal bones. Furthermore, light- and electron-microscopic examinations revealed that bone mineralization proceeded through formation of matrix vesicles, without any metastatic or dystrophic calcification. These in vitro findings suggest that 17 beta-estradiol elicits small, but reproducible, direct effects on calcium content in the parietal bones not only in female but also in male fetal mice at physiological-free E2 concentrations (10(-12)-10(-11) mol/L), which is attainable in serum of normal human subjects. In contrast to in vivo studies, pharmacological doses of 17 beta-estradiol had no anabolic effect on parietal bones. The mechanism of such a biphasic effect of estrogens remains to be elucidated.

Oestrogen and insulin interact at the level of transcription in breast cancer cells.

‘the growth of the human breast cancer cell line, MCF-7, is stimulated by oestrogen and inhibited by antioestrogens both in vitro and in vivo [1,2]. MCF-7 cells may therefore serve as a model of those human breast tumours that are responsive to endocrine therapy. In addition to oestrogen receptors, MCF-7 cells possess receptors for, and respond to, a wide range of other hormones and growth factors [3]. Insulin is a mitogen for MCF-7 cells and synergises with oestradiol in stimulating cell growth [4]. We have investigated the interaction between insulin and oestradiol in regulating the expression of two oestrogeninducible genes, LIV-1 [5] and pS2 [6], in MCF-7 cells. MCF-7 cells were withdrawn from oestradiol by culturing them in phenol red-free medium, supplemented with dextrad charcoal treated serum, for 6 days. Withdrawn cells were then cultured in medium containing oestradiol(10-8111) with or without insulin (lpg/ml). Cells were harvested after 3 ,6 ,24 or 48 hours exposure to the hormondagents. LIV-1 and pS2 mRNA levels were determined by Northern blot [7]. The results are expressed as fold increase relative to the mRNA levels detected in untreated (control) cells. Nuclei were isolated from cells and the rates of LIV-1 and pS2 transcription were assayed using the run-on, in vitro transcription technique [7]. The resultant autoradiographs were quantified by densitometry and the LIV-1 and pS2 results were normalised relative to the actin (uninduced control) result. Results are expressed as fold increase with respect to the rate of transcription in untreated (control) cells. No hybridisation to the negative controls (rhodopsin, pBR322, pBluescript) was detected. Figure 1 presents the results from three such experiments. LIV1 and pS2 mRNA levels show a maximum increase in response to oestradiol and to oestradiol+insulin at 6hrs after hormone addition (Fig. l(a)). LIV1 mRNA was induced 1 1.3 fold by oestradiol alone and 26.1 fold by oestradiol+insulin. pS2 mRNA was induced 14.1 fold by oestradiol and 29.4 fold by oestradiol+insuhn. The mRNA levels declined only slowly from their 6hr maximum and remained at >70% of maximum by 48hrs. At all time points the oestradiol+insulin mRNA levels were 1.5 2.0 times that seen with oestradiol alone. It should be noted that the concentration of oestradiol used (10-8M) induces an optimal increase in LIV-1 and pS2, in the absence of insulin [7]. Evidently, induction by insulin occurs by a different but complementary mechanism to that of oestradiol. When the rate of LIV-1 and pS2 transcription was determined it was Seen that a maximal increase was reached by 3hrs after hormone addition (Fig.l(b)). LIV-1 transcription exhibited a 6.1 ( 9 . 5 ) fold increase in response to oestradiol and a 13.7 (9.4) fold increase in response to oestradiol+insulin. pS2 transcription was stimulated 8.6 (9.3) fold by oestradiol and 19.9 h0.3) fold by oestradiol+insulin. Transcription rate declined more rapidly from its 3 hr rate and by 24 hrs was <15% of maximum, in all cases. The more prolonged elevation of the mRNA levels must therefore reflect the stability of the messenger RNAs rather than their continued synthesis. Once again, insulin is able to produce a significantly (p< 0.001) greater increase in transcription than that induced by the optimal concentration of oestradiol alone. Our results show that the relatively long-lasting induction of LIV1 and pS2 mRNA by oestradiol and by oestradiol+insulin, results from a much briefer alteration in the rate of gene transcription. The reson for the rapid decline in transcription rate after 3 hrs is unclear but presumably results from a decrease in the amount or activity of the oestrogen receptor [9]. The different but complementary mechanisms of transcriptional stimulation used by oestradiol and insulin both require the oestrogen receptor. This example of cross-talk between two very different intracellular signalling systems is of interest in its own 30

Estrogen suppresses stromal cell-dependent lymphopoiesis in culture.

Numbers of pre-B cells change dramatically and reciprocally in response to estrogen levels in mice, suggesting that normal lymphopoiesis may be under hormonal control. However, little is known of the mechanisms involved in this process. We found that estrogen receptor mRNA was detectable by RT-PCR in lymphocyte supporting stromal cells as well as B lymphocyte precursors. Unlike glucocorticoids, estrogen did not induce apoptosis in isolated B lineage lymphocytes or interfere with their responsiveness to IL-7 in semisolid agar. Estrogen did inhibit clonal expansion of B cell precursors in a limiting dilution-type assay when the lymphocytes were cultured on a stromal cell clone. In other experiments, B cell precursors at particular stages of differentiation were isolated by cell sorting and cocultured with stromal cells for 4 days. This revealed that some subsets were more sensitive to an estrogen-containing environment than others. Although numbers of recovered cells were greatly reduced, the remaining lymphocytes had undergone relatively normal differentiation. The surviving population was enriched in cells that had acquired cytoplasmic mu chains, BP-1 Ag, and clonability with IL-7. Hormone-mediated inhibition occurred in serum and phenol-red free medium, and in cultures replete with IL-7. Direct contact between stromal cells and lymphocytes was not required. Furthermore, suppression resulted when stromal cells alone were treated with the hormone. These findings indicate that estrogen may regulate B lymphopoiesis via its influence on the microenvironment and that estrogen-induced stromal cell genes merit further study.

A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins

A number of proteins have been rendered functionally oestrogen-dependent by fusion with the hormone-binding domain of the oestrogen receptor. There are, however, several significant disadvantages with such fusion proteins. First, their use in cells in vitro requires phenol red-free medium and laborious stripping of steroid hormones from serum in order to avoid constitutive activation. Secondly, control of oestrogen receptor fusion proteins in vivo is precluded by high endogenous levels of circulating oestrogens. Thirdly, the hormone-binding domain of the oestrogen receptor functions as a hormone-dependent transcriptional activation domain making interpretation of fusions with transcription factors problematical. In order to overcome these drawbacks we have used a transcriptionally inactive mutant of the murine oestrogen receptor which is unable to bind oestrogen yet retains normal affinity for the synthetic ligand, 4-hydroxytamoxifen. When the hormone-binding domain of this mutant oestrogen receptor is fused to the C-terminus of the c-Myc protein, Myc-induced proliferation and apoptosis in fibroblasts becomes dependent on 4-hydroxytamoxifen, but remains refractory to 17 beta-oestradiol.

Modulation by Progesterone of Interleukin‐6 Production by Gingival Fibroblasts

The gingivitis associated with pregnancy has been attributed to increased concentrations of circulating estrogen and/or progesterone. However, the mechanism by which these steroids increase gingival inflammation is not known. Interleukin-6 (IL-6), a pleiotropic cytokine produced by many cell types including human gingival fibroblasts (hGF), is secreted in response to inflammatory challenges such as bacterial lipopolysaccharide and interleukin-1 (IL-1). This study tested the hypothesis that progesterone could modulate the local production of IL-6 by hGF. The effects of progesterone on IL-6 production were measured in vitro in serum-free, phenol red-free medium to eliminate possible effects of such medium additives. The concentration of IL-6 secreted into supernatant medium after a 24 hour challenge with IL-1 beta was estimated by radioimmunoassay. Total RNA from steroid-treated hGF was probed for IL-6 mRNA. In serum-free medium, progesterone dose-dependently and significantly (P < 0.05) inhibited IL-6 production by hGF, as did the glucocorticoids hydrocortisone (HC) and dexamethasone. At progesterone concentrations common in late pregnancy, IL-6 production was reduced to levels 40 to 50% of control. In addition, mRNA was significantly down-regulated by progesterone and HC, at both basal levels and after IL-1 beta challenge. These results suggest that high levels of progesterone during pregnancy affect the development of localized inflammation by down-regulation of IL-6 production, rendering the gingiva less efficient at resisting the inflammatory challenges produced by bacteria.

THE IN-VITRO RESPONSE OF 4 ANTISTEROID RECEPTOR AGENTS ON THE HORMONE-RESPONSIVE PROSTATE-CANCER CELL-LINE LNCAP

Previous reports indicate that flutamide withdrawal is associated with PSA declines and tumor shrinkage in selected patients with 'hormone-refractory' prostate cancer. Though the mechanisms underlying this effect are not clear, investigators have hypothesized that these effects are mediated by mutant androgen receptors recognizing hydroxy-flutamide as an androgenic agonist. Such receptors have been well described in the human prostate cancer cell line LNCaP. Despite the finding that the androgen receptor of LNCaP aberrantly recognizes a variety of steroids, including estrogen and progesterone, as androgenic agonists, there are no studies which examine the effect of estrogen antagonists and progesterone antagonist on baseline and androgen-stimulated LNCaP growth. In this report, LNCaP cells were cultured in phenol red-free media using charcoal-stripped sera. As previously reported, flutamide enhanced LNCaP growth and bicalutamide inhibited androgen-stimulated LNCaP proliferation. Neither tamoxifen nor RU486 influenced LNCaP growth (either in the presence or absence of exogenous androgens). From these data we conclude that antagonists of estrogen and progesterone action have no anti-proliferative effect on LNCaP cells and that the mutant androgen receptor expressed in these cells is quite restrictive in the recognition of compounds with antagonistic activity. The clinical implications of these findings are discussed.

Effects of oestradiol and tamoxifen on oxytocin-induced phospholipase C activation in human myometrial cells

The role of oestradiol in the control of uterine responsiveness to oxytocin was investigated by measuring oxytocin-induced phospholipase C activation in [3H]inositol-labelled cultured human myometrial cells. Addition of oestradiol to steroid-free culture medium (10% (v/v) fetal calf serum treated with dextran-coated charcoal in phenol red-free medium) enhanced formation of inositol phosphates and this effect was completely abolished by the anti-oestrogen tamoxifen. The inhibitory effect of tamoxifen on oxytocin-induced phospholipase C activation occurred in both steroid-free and complete culture medium; it was time- and concentration-dependent and was only partly reversed by oestradiol. When phospholipase C was activated with PGF2 alpha or fluoroaluminate instead of oxytocin, oestradiol and tamoxifen had the same stimulatory and inhibitory effects, respectively. The inhibitory effect of tamoxifen could not be prevented by treating the cells with pertussis toxin. Moreover, the effect of tamoxifen was not mediated by inhibition of protein kinase C, since the use of staurosporine (a protein kinase inhibitor) resulted in potentiation of phospholipase C activation by oxytocin. Both oestradiol and tamoxifen increased [3H]inositol incorporation into cellular lipids and cell proliferation. These results suggest that oestradiol enhances myometrial responsiveness to oxytocin and other agonists by facilitating phospholipase C activation at a post-receptor level. This effect is antagonized by tamoxifen; however, tamoxifen also has oestrogen-independent inhibitory effects.