Cells Express Estrogen Receptors Research Articles

Estrogen receptor expression in meibomian glands and its correlation with age and dry-eye parameters

To investigate the correlation between estrogen receptor-positive basal cells of the meibomian glands of the lower lid with age, gender, and subjective and objective dry-eye parameters. Sixteen lower lid specimens were collected from 7 female and 9 male patients (age range 22-88 years) during tumor surgery requiring whole-thickness excision. Prior to surgery the patients were asked about any dry-eye symptoms (score 0-4, where 0=no dry-eye symptoms). Tear break-up time was measured, and Schirmer I and II tests were performed. We obtained histological sections from the outer margins of the formalin-fixed and paraffin-embedded specimens and performed immunohistochemical staining for estrogen, progesterone and androgen receptors. At least 500 cells were counted per specimen, and the proportion of positively stained cells was calculated and correlated to the age and dry-eye parameters. All meibomian glands had positive nuclear staining with antibodies for estrogen receptors in their outer cell layers, i.e., the basal cell layer. The proportion of cells expressing estrogen receptors increased with age independent of gender (correlation coefficient=0.67, P<0.005). No correlation was found between estrogen receptor positivity and subjective dry-eye symptoms, tear break-up time, or Schirmer I and II test results. There was no difference in the proportion of cells expressing estrogen receptors between female and male patients (P=0.5). The amount of meibomian gland cells expressing estrogen receptors in the lower lid seems to increase with age independent of gender and seems not to affect the fat layer and stability of the tear film.

Characterization of reproductive steroid receptors and response to estrogen in a rat serotonergic cell line

Study of the cellular and molecular consequences of steroid hormone action in the serotonin neural system will provide new avenues for pharmacotherapeutic intervention in mental illness related to reproductive function. However, it is difficult to probe intracellular mechanisms with whole animal models. We sought the steroid receptor compliment and estrogen response of two rat serotonin cell lines in order to determine if they could be of future assistance in this matter. Immunohistochemistry with a panel of antibodies, RT-PCR and a serotonin ELISA were utilized to characterize the RN46A-V1 cells (herein called RN46A), and the subclone RN46A-B14 (herein called B14) that is stably transfected with brain derived neurotrophic factor (BDNF). RN46A and B14 cells express estrogen receptor beta (ERβ), androgen receptors (AR) and nuclear factor kappa B (NFκB) but not estrogen receptor alpha (ERα) or progestin receptors (PR). RT-PCR confirmed the presence of ERβ and the absence of ERα and PR in both cell lines. B14 cells contain more immunodetectable BDNF and serotonin than the RN46A parent line. In addition, immunofluorescence for the serotonin reuptake transporter (SERT) was observed in the cell body region of undifferentiated B14 cells. After differentiation at a nonpermissive temperature, SERT immunostaining was observed in both the cell body region and along the extent of the axons. Serotonin content as determined by ELISA was higher in B14 than RN46A cells. Estrogen (0.1 and 1.0 nM) stimulated serotonin in the B14 cells in serum free medium. In summary, the RN46A cells and the B14 subclone contain the same compliment of nuclear steroid receptors as rat raphe serotonin neurons and thus may provide a convenient in vitro model for study of intracellular mechanisms of action of steroid hormones in the context of a serotonin neuron.

Gonadectomy induces site-specific differences in nociception in rats

High prevalence of chronic orofacial pain in women and its relationship with ovarian states suggest that ovarian hormones may be involved in the control of orofacial nociception. Since the interaction between ovarian hormones and nociception seems more evident in the orofacial area than in many other parts of the body, a possible site specificity of an ovarian hormone effect on nociception was tested in rats. Two nociceptive tests were applied to three groups of male rats ( n=46) and three groups of female rats ( n=46), that were gonadectomised ( n=17), sham-operated ( n=15) or intact ( n=14). Each rat in each group received a local subcutaneous injection of formalin in the upper lip and in the hindpaw. Upper lip injection resulted in an increased occurrence of upper lip rubbing for more than 45 min and hindpaw injection resulted in an increased occurrence of hindpaw licking for about 1 h. The duration of the nociceptive behaviours was measured at 3 months after surgery. No significant difference was found between intact and sham-operated animals. A significant increase (54%) in the upper lip rubbing but not the hindpaw licking was observed in gonadectomised females. No difference was observed in castrated males for upper lip rubbing, but a tendency towards an increased duration (102%) of hindpaw licking was noted. The depletion in gonadal hormones was confirmed 3 months after gonadectomy and after the sacrifice of the animals, by the observed decline in the bone mineral density measured on the femur of 40 rats belonging to the six groups. A role of ovarian hormones was also suggested after immunostaining of oestrogen receptors in the lamina II of Caudalis subnucleus of the trigeminal sensory complex and cervical (C1–C2) spinal dorsal horn. The number of cells expressing oestrogen receptors displayed a small (13.6%) but significant ( P=0.037) increase in ovariectomised compared with sham-operated rats. These results suggest that the lack of ovarian hormones induces a site-specific increase in the sensitivity to orofacial nociceptive stimulation, and that an up-regulation of oestrogen receptors in the Caudalis subnucleus and C1-C2 dorsal horn may be one of the factors involved in this effect.

Altered mammary gland differentiation and progesterone receptor expression in rats fed soy and whey proteins.

There are suspected links between an animal's diet, differentiation status of a target tissue, and sensitivity to chemically induced cancer. We have demonstrated that rats fed AIN93G diets made with soy protein isolate (SPI) or whey protein hydrolysate (WPH) had a lower incidence of 7,12-dimethylbenz(a)anthracene (DMBA)-induced adenocarcinoma than rats fed the same diet made with casein (CAS). The current study was conducted to determine the differentiation status of the mammary glands during development. Offspring of rats (n = 5-10/group) were fed diets made with SPI, WPH, or CAS throughout life (beginning on gestation day 4) and were sacrificed on postnatal day (PND) 21, PND 33, PND 50 or on metaestrous between PND 48 and PND 51. There were no significant differences between the numbers of mammary terminal end buds (TEBs) or lobuloalveoli (LOB) between any of the diets groups at PND 21 or PND 33, but at PND 50 there was an 75% decrease in the mean numbers of TEBs/mm(2) in the SPI- or WPH-fed rats, compared with the CAS-fed rats (p = 0.09 and p = 0.06, respectively). In rats sacrificed in metaestrous, there were no significant differences in the proliferation index (PI) in the TEBs or LOB between any of the diet groups. In metaestrous rats, there were twice as many cells expressing estrogen receptor beta (ERbeta; approximately 60%) compared with estrogen receptor alpha (ERalpha; approximately 30%) in the LOB and 1.5 times more ERbeta (approximately 60%) compared with estrogen receptor alpha (ERalpha, approximately 40%) in the TEBs. There were no diet-dependent differences in expression of ERalpha and ERbeta. Similarly, there were no differences between the diet groups in progesterone receptor (PR) expressing LOB cells. However, in the TEBs there was a diet-dependent difference in PR positive cells with a 34% increase (p < 0.05) in the SPI-fed rats and a 38% increase (p < 0.05) in the WPH-fed rats compared with the CAS-fed rats. These results show that the type of dietary protein alters the phenotype of mammary epithelia in the TEBs. The SPI- and WPH-dependent changes in mammary differentiation may contribute to the reduced sensitivity to DMBA-induced mammary cancer in rats fed these proteins.

Zur Hormonsubstitution beim malignen Melanom

Beim malignen Melanom handelt es sich um einen Tumor, der von den pigmentbildenden Zellen der Haut ausgeht. Melanomzellen exprimieren teilweise und in unterschiedlichem Ausmaß Estrogenrezeptoren. In tierexperimentellen Untersuchungen konnte gezeigt werden, dass Sexualhormone einen Einfluss auf das Wachstumsverhalten von Melanomzellen ausüben. Es stellt sich daher die Frage, ob bei Frauen mit Melanomerkrankung die Zufuhr exogener Sexualhormone zulässig oder kontraindiziert ist.

Expression and regulation of WNT10B in human cancer: Up-regulation of WNT10B in MCF-7 cells by β-estradiol and down-regulation of WNT10B in NT2 cells by retinoic acid

WNT signaling molecules, playing key roles in embryogenesis and carcinogenesis, are potent targets for regenerative medicine and clinical oncology. We have previously cloned and characterized the human orthologue of mouse proto-oncogene Wnt-10b using bioinformatics and cDNA-PCR. Human WNT10B is moderately expressed in MKN45 and MKN74 cells derived from human gastric cancer, and is up-regulated by tumor necrosis factor alpha (TNFalpha) in MKN45 cells. Here, expression and regulation of WNT10B in human cancer other than gastric cancer were investigated using cDNA-PCR. WNT10B mRNA was expressed in the majority of squamous cell carcinoma cell lines derived from esophageal cancer and cervical cancer. WNT10B mRNA was relatively highly expressed in TE3, TE6, TE10, TE11 (esophageal cancer), Hs700T (pancreatic cancer), SKG-IIIa, HeLa S3 (cervical cancer), and T-47D (breast cancer). Expression of WNT10B mRNA was up-regulated by beta-estradiol in MCF-7 cells expressing estrogen receptors. Expression of WNT10B mRNA was down-regulated by all-trans retinoic acid in NT2 cells with the potential of self renewal and neuronal differentiation. WNT10B might be implicated in self renewal of stem cells as well as in carcinogenesis through activation of the WNT - beta-catenin pathway.

Baicalein and genistein display differential actions on estrogen receptor (ER) transactivation and apoptosis in MCF-7 cells

Genistein is a soy isoflavone that has attracted much attention for its chemopreventive properties. Baicalein is a flavone found in Scutellaria species, and its structure is analogous to that of genistein. We examined the two flavones’ antiestrogenicity by using a transient transfection model. Our results indicated that baicalein could suppress 17β-estradiol-induced transactivation in cells expressing estrogen receptor α, whereas no antagonistic activity was seen in genistein. Cell death assay and flow cytometry analysis demonstrated that baicalein was a stronger apoptosis inducing agent than genistein. Considering all the criteria tested, baicalein could be a better chemopreventive agent than genistein.

Induction of Endothelial Nitric-oxide Synthase Phosphorylation by the Raloxifene Analog LY117018 Is Differentially Mediated by Akt and Extracellular Signal-regulated Protein Kinase in Vascular Endothelial Cells

Raloxifene is a tissue-selective estrogen receptor modulator. The effect of estrogen on cardiovascular disease is mainly dependent on direct actions on the vascular wall involving activation of endothelial nitric oxide synthase (eNOS) via Akt and extracellular signal-regulated protein kinase (ERK) cascades. Although raloxifene is also known to activate eNOS in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), the raloxifene analog LY117018 caused acute phosphorylation of eNOS that was unaffected by actinomycin D and was blocked by the pure estrogen receptor antagonist ICI182,780. Activation of Akt by raloxifene reached a plateau at 15-30 min and declined thereafter, a similar time frame to that of Akt activation by 17beta-estradiol. On the other hand, both activation and phosphorylation of ERK by raloxifene showed a biphasic pattern (peaks at 5 min and 1 h), whereas ERK activation and phosphorylation by 17beta-estradiol reached a plateau at 5 min and declined thereafter. A MEK inhibitor, PD98059, had no effect on the raloxifene-induced Akt activity, suggesting an absence of cross-talk between the ERK and Akt cascades. Either exogenous expression of a dominant-negative Akt or pretreatment of TRLECs with PD98059 decreased the raloxifene-induced eNOS phosphorylation. Moreover, raloxifene stimulated the activation of Akt, ERK, and eNOS in Chinese hamster ovary cells expressing estrogen receptor alpha but not Chinese hamster ovary cells expressing estrogen receptor beta. Our findings suggest that raloxifene-induced eNOS phosphorylation is mediated by estrogen receptor alpha via a nongenomic mechanism and is differentially mediated by Akt- and ERK-dependent cascades.

Vascular nitric oxide, sex hormone replacement, and fish oil may help to prevent Alzheimer's disease by suppressing synthesis of acute-phase cytokines

The neurodegenerative plaques of Alzheimer's disease (AD) are characterized by a self-sustaining acute-phase reaction in which both interleukin-1 (IL-1) and interleukin-6 (IL-6) are up-regulated. The fact that IL-6 is detectable in early stage diffuse plaques encourages the speculation that the acute-phase process is crucial to the pathogenesis of AD. The epidemiological association of AD with estrogen deficiency, as well as with various disorders characterized by vascular endotheliopathy, suggest a protective role for vascular nitric oxide (NO). NO has an autocrine anti-inflammatory impact on endothelium, owing in part to antagonism of NF-κB activity; since induction of IL-6 is dependent on NF-κB, this may explain recent evidence that NOinhibits macrophage IL-6 production. It is reasonable to postulate that, analogously, cerebrovascular NOdecreases IL-6 production in the brain. Vascular NO may also have direct neuroprotective activity. Estrogen, in addition to promoting vascular NO synthesis, can block IL-6 production by a more direct mechanism in cells expressing estrogen receptors; since such receptors have been reported in brain glia and astrocytes, estrogen has the potential to limit brain IL-1 activity. Testosterone likewise can inhibit IL-6 induction in androgen-responsive cells, which may include brain glia and astrocytes. Since fish oil and γ linolenic acid (GLA) suppress IL-1 production by stimulated monocytes, they conceivably could exert this effect in the brain as well; the comparatively low prevalence of AD in elderly Japanese is intriguing in this regard. These considerations suggest that a healthy cerebrovascular endothelium, sex hormone activity, and dietary fish oil/GLA may slow or prevent AD onset by dampening acute-phase mechanisms in the brain.

Apoptosis and Tumorigenesis in Human Cholangiocarcinoma Cells: Involvement of Fas/APO-1 (CD95) and Calmodulin

We have previously demonstrated that tamoxifen inhibits the growth of human cholangiocarcinoma cells in culture and inhibits tumor growth when cells are injected into nude mice. However, the mechanism of action of tamoxifen remains unknown. Here we demonstrate that tamoxifen and trifluoperazine, both potent calmodulin antagonists, induce apoptosis in vitro, probably acting via the Fas system, in human cholangiocarcinoma cells. Human cholangiocarcinoma cell lines heterogeneously express Fas antigen on their surface. Fas-negative and Fas-positive surface-expressing cells were isolated, cloned, and cultured. Fas antibody, tamoxifen, and trifluoperazine induced dose-dependent apoptosis only in Fas-positive cells; Fas-negative cells were unaffected. Furthermore, apoptosis induced by tamoxifen in Fas-positive cells was blocked by an inhibitory Fas antibody. Tamoxifen was not acting through an anti-estrogenic mechanism, because neither Fas-negative nor Fas-positive cells expressed estrogen receptors and the pure anti-estrogen compound, ICI 182780, did not induce apoptosis in either cell line. Fas-negative cells, but not Fas-positive cells, were able to produce tumors when subcutaneously injected into nude mice. These findings suggest Fas may be a candidate oncogene involved in the pathogenesis of cholangiocarcinoma. Furthermore, the similarity between the pro-apoptotic effects of tamoxifen and trifluoperazine support an underlying molecular mechanism for Fas-mediated apoptosis that involves calmodulin.

Endometrial Endothelial Cells Express Estrogen and Progesterone Receptors and Exhibit a Tissue Specific Response to Angiogenic Growth Factors

To develop a reliable method for the isolation and longterm culture of microvessel endothelial cells from human endometrium and to evaluate their response to angiogenic growth factors and steroid hormones in comparison to endothelial cells derived from other organs. Endometrial tissue from hysterectomy specimens were digested sequentially with collagenase and trypsin, cultured for 24 h, then selected by adhesion to anti-CD-34 coated magnetic beads. Alternatively, anti-CD-34-coated beads could also be substituted by Ulex europaeus agglutinin-1, anti-PECAM, or anti-E-selectin-coated beads. Characterization of the isolated cultures included expression of endothelial cell markers, regulation of E-selectin in response to TNF-alpha, proliferative response to angiogenic growth factors, and expression of progesterone and estrogen receptors. We also analyzed the relative binding affinity of VEGF on endometrial endothelial cells in comparison to other endothelial cell types. Selection on anti-CD-34-coated beads eliminated contaminating cells and resulted in a homogeneous population of human endometrial endothelial cells (HEEC), as assessed by expression of PECAM, von Willebrand's factor, and uptake of acetylated-LDL. HEEC also upregulated E-selectin in response to TNF-alpha in a manner similar to that seen for other endothelial cell types. Expression of progesterone and estrogen receptor was revealed by immunocytochemistry and RT-PCR consistently until passage 5. Endometrial endothelial cells were more responsive to growth stimulation by VEGF than were dermal endothelial cells isolated under similar conditions. Further characterization indicated that VEGF bound more avidly to HEEC than to other endothelial cell types. Human endometrial endothelial cells were isolated to homogeneity by a two-part protocol and successfully passaged under culture conditions similar to those used for other endothelial cell types. The HEEC were very responsive to VEGF growth-stimulation likely due to elevated affinity, or increased levels of, KDR and FLT-1 on the cell surface. These results indicate that HEEC are capable of maintaining a mature phenotype in culture and might provide a model for understanding the response of these cells to the recurrent cycles of proliferation imposed on the endometrium during menstruation.

Endometrial Endothelial Cells Express Estrogen and Progesterone Receptors and Exhibit a Tissue Specific Response to Angiogenic Growth Factors

Endometrial Endothelial Cells Express Estrogen and Progesterone Receptors and Exhibit a Tissue Specific Response to Angiogenic Growth Factors

Endometrial Endothelial Cells Express Estrogen and Progesterone Receptors and Exhibit a Tissue Specific Response to Angiogenic Growth Factors

Endometrial Endothelial Cells Express Estrogen and Progesterone Receptors and Exhibit a Tissue Specific Response to Angiogenic Growth Factors

Characterization of a cell line established from diethylstilbestrol-induced renal tumors in Syrian hamsters.

This article describes HKT-1097, a new cell line established from renal tumors induced by the protracted administration of diethylstilbestrol (DES) to male Syrian golden hamsters. Cell culture was initiated from tumor samples obtained from two 14-mo.-old animals which had undergone exposure to DES for a period of 11 mo. The HKT-1097 cell line was characterized between Passages 16 and 22 with respect to cell morphology, growth properties, karyology, and the presence of estrogen receptors. Moreover, immunostaining with a panel of antisera was performed to identify the cytological profile of the cell line and establish a parallel with tumor tissue in vivo. HKT-1097 cells are fibroblastoid; their most distinctive feature is that they exhibit strikingly long processes. The HKT-1097 cell line grows as a monolayer with a tendency toward a less stringent density-dependent inhibition of growth. The modal chromosome number is 44, but more than 50% of the cells are aneuploid, suggesting a substantial degree of karyotype instability. HKT-1097 cells express estrogen receptors. They contain immunoreactive vimentin and desmin, but appear negative upon cytokeratin immunostaining. In addition, these cells express glial fibrillary acidic protein and other markers of the neuroectodermal lineage, but lack neurofilament protein. Insofar as the same lineage markers have been demonstrated in DES-induced Syrian hamster kidney tumors (SHKT), we conclude that HKT-1097 cells retain some of the original tumor cell phenotype. The current observations suggest that estrogen-induced SHKT derive from the renal interstitium and point to an involvement of neuroectodermal cells in the development of these neoplasms.

Estrogen and aryl hydrocarbon responsiveness of ECC-1 endometrial cancer cells

ECC-1 endometrial cancer cells express estrogen receptor α (ER α), and 17β-estradiol (E2) induces cell proliferation, cathepsin D mRNA levels, and reporter gene activity in cells transiently transfected with constructs derived from the human cathepsin D and creatine kinase B (pCD and pCKB, respectively) gene promoters. The comparative antiestrogenic activity of aryl hydrocarbon receptor (AhR) agonists and ER α antagonists were also determined in these endometrial cancer cells. A functional AhR was expressed in ECC-1 cells and AhR agonists including 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) inhibited E2-induced cell proliferation and transactivation. This was comparable to inhibitory AhR-ER crosstalk in breast cancer cell lines. The pure ER antagonist ICI 182,780 also exhibited antiestrogenic activity in ECC-1 cells; however, the results obtained for 4′-hydroxytamoxifen were response-specific. 4′-Hydroxytamoxifen alone did not induce ECC-1 cell proliferation but completely inhibited E2-induced cell proliferation. 4′-Hydroxytamoxifen primarily exhibited ER antagonist activities in transactivation assays and this contrasted to the predominant ER agonist responses observed in other endometrial cancer cell lines. The unique cellular context of ECC-1 cells was confirmed using pCKB and constructs expressing wild-type ER or ER variants expressing activation function 1 (AF1) or AF2 (ER-AF1 and ER-AF2, respectively). 4′-Hydroxytamoxifen did not induce reporter gene activity in cells cotransfected with pCKB and ER-AF1 or ER-AF2; however, in cotreatment studies (4′-hydroxytamoxifen plus E2), 4′-hydroxytamoxifen inhibited E2-induced transcriptional activation by ER-AF1 or ER-AF2. Thus, the primarily antiestrogenic activity observed for 4′-hydroxytamoxifen in ECC-1 cells may be related to the inability to activate gene expression through AF1-dependent pathways.

Endometrial Endothelial Cells Express Estrogen and Progesterone Receptors and Exhibit a Tissue Specific Response to Angiogenic Growth Factors

Endometrial Endothelial Cells Express Estrogen and Progesterone Receptors and Exhibit a Tissue Specific Response to Angiogenic Growth Factors

The Activation of Plasminogen Activator Inhibitor-1 Expression by IL-1β Is Attenuated by Estrogen in Hepatoblastoma HepG2 Cells Expressing Estrogen Receptor α

A low estrogen status in postmenopausal women is associated with elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1). In this study, the ability of estrogen compounds to regulate PAI-1 expression was determined in a hepatocyte HepG2 cell line made to stably express estrogen receptor alpha (ERalpha). In both the wild type and ER expressing HepG2 cells, estrogen had no effect on basal PAI-1 expression. However, in the ER expressing cells the ability of IL-1beta to increase PAI-1 mRNA and protein levels was attenuated by 17beta-estradiol, tamoxifen and twelve estrogen components of Premarin. In contrast, the mixed agonist/antagonist raloxifene had weak agonist activity and like the pure antagonist ICI 182780, it dose dependently blocked the effect of 17beta-estradiol on IL-1beta stimulated PAI-1 levels. These results suggest that estrogen agonists may lower PAI-1 levels in vivo by inhibiting cytokine activated PAI-1 expression by an ER dependent mechanism.

Anti-Estrogens Induce Apoptosis of Multiple Myeloma Cells

Previous studies have suggested that multiple myeloma (MM) cells express estrogen receptors (ER). In the present study, we characterized the effects of estrogen agonists and antagonists (anti-estrogens [AE]) on growth of MM cell lines and MM patient cells. In addition to antagonizing estrogen binding to ER, AE can trigger apoptosis. Hence, we also determined whether estrogens or AE altered MM cell survival. Immunoblotting showed that ER- is expressed in 4 of 5 MM cell lines (ARH-77, RPMI 8226, S6B45, and U266, but not OCI-My-5 cells), as well as in freshly isolated MM cells from 3 of 3 patients. 17β-estradiol (E2) did not significantly alter proliferation of MM cell lines or MM patient cells. In contrast, two structurally distinct AE, tamoxifen (TAM) and ICI 182,780 (ICI), significantly inhibited the proliferation of all 5 MM cell lines and MM cells from 2 of 2 patients (IC50, 2 to 4 μmol/L). Proliferation of these cell lines was also inhibited by the hydroxylated TAM derivative, 4-hydroxytamoxifen (4HTAM), although this derivative was less potent than TAM (IC50, 3 to 25 μmol/L). In contrast, the dehalogenated TAM derivative toremifene (TOR) did not inhibit MM cell proliferation. We next examined the effects of these agents on MM cell survival. TAM, ICI, and, to a lesser extent, 4HTAM and TOR triggered apoptosis in both ER-–positive as well as ER-–negative MM cell lines and patient MM cells, evidenced both by fluorescence-activated cell sorting (FACS) analysis using propidium iodide staining and the TUNEL assay. TAM-induced growth inhibition and apoptosis of ER-–positive S6B45 MM cells was not blocked by coculture with excess E2. TAM-induced apoptosis of S6B45 MM cells was also unaffected by addition of exogenous interleukin-6. Importantly, both the inhibition of MM cell proliferation and the induction of MM cell apoptosis were achieved at concentrations of TAM (0.5 and 5.0 μmol/L) that did not significantly alter in vitro growth of normal hematopoietic progenitor cells. Similar plasma levels of TAM have been achieved using high-dose oral TAM therapy, with an acceptable toxicity profile. These studies therefore provide the rationale for trials to define the utility of AE therapy in MM.© 1998 by The American Society of Hematology.

Anti-Estrogens Induce Apoptosis of Multiple Myeloma Cells

Previous studies have suggested that multiple myeloma (MM) cells express estrogen receptors (ER). In the present study, we characterized the effects of estrogen agonists and antagonists (anti-estrogens [AE]) on growth of MM cell lines and MM patient cells. In addition to antagonizing estrogen binding to ER, AE can trigger apoptosis. Hence, we also determined whether estrogens or AE altered MM cell survival. Immunoblotting showed that ER-α is expressed in 4 of 5 MM cell lines (ARH-77, RPMI 8226, S6B45, and U266, but not OCI-My-5 cells), as well as in freshly isolated MM cells from 3 of 3 patients. 17β-estradiol (E2) did not significantly alter proliferation of MM cell lines or MM patient cells. In contrast, two structurally distinct AE, tamoxifen (TAM) and ICI 182,780 (ICI), significantly inhibited the proliferation of all 5 MM cell lines and MM cells from 2 of 2 patients (IC50, 2 to 4 μmol/L). Proliferation of these cell lines was also inhibited by the hydroxylated TAM derivative, 4-hydroxytamoxifen (4HTAM), although this derivative was less potent than TAM (IC50, 3 to 25 μmol/L). In contrast, the dehalogenated TAM derivative toremifene (TOR) did not inhibit MM cell proliferation. We next examined the effects of these agents on MM cell survival. TAM, ICI, and, to a lesser extent, 4HTAM and TOR triggered apoptosis in both ER-α–positive as well as ER-α–negative MM cell lines and patient MM cells, evidenced both by fluorescence-activated cell sorting (FACS) analysis using propidium iodide staining and the TUNEL assay. TAM-induced growth inhibition and apoptosis of ER-α–positive S6B45 MM cells was not blocked by coculture with excess E2. TAM-induced apoptosis of S6B45 MM cells was also unaffected by addition of exogenous interleukin-6. Importantly, both the inhibition of MM cell proliferation and the induction of MM cell apoptosis were achieved at concentrations of TAM (0.5 and 5.0 μmol/L) that did not significantly alter in vitro growth of normal hematopoietic progenitor cells. Similar plasma levels of TAM have been achieved using high-dose oral TAM therapy, with an acceptable toxicity profile. These studies therefore provide the rationale for trials to define the utility of AE therapy in MM.© 1998 by The American Society of Hematology.

Estrogen stimulates delayed mitogen-activated protein kinase activity in human endothelial cells via an autocrine loop that involves basic fibroblast growth factor.

Estrogen plays a significant role in protecting premenopausal women from cardiovascular disease. We have found that estradiol augments endothelial cell activities related to vascular healing and that human coronary artery and umbilical vein endothelial cells express estrogen receptors (ERs). Classically, the ER functions as a transcription factor, but the cytoplasmic targets of this genomic effect have not been defined for endothelial cells. In the present study, we examined the potential role of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 as mediators of estrogen action. Human umbilical vein endothelial cells were estrogen depleted by culturing in hormone-free medium for 48 hours before experiments. 17Beta-estradiol (E2) stimulated a delayed (3 hours) 5- to 7-fold induction of ERK1/2 activity requiring activation of ER and new transcription/translation. Conditioned media from cells stimulated for 3 hours with E2 induced immediate ERK1/2 activation and phosphorylation of the basic fibroblast growth factor (bFGF) receptor. Moreover, ERK1/2 activation by E2 or by conditioned media was abrogated by treatment with neutralizing anti-bFGF antibody. These data describe an autocrine mechanism for E2 induction of ERK1/2 in HUVEC. Because our previous studies suggested that certain cardioprotective effects of estrogen are genomic in nature, the results are consistent with the hypothesis that autocrine stimulation of endothelial ERK1/2 activity by bFGF may play a role in the beneficial effects of estrogen on cardiovascular biology.