Dietary soy phytoestrogens and ERalpha signalling modulate interferon gamma production in response to bacterial infection.

Diets rich in soy phytoestrogens have many potential health benefits but isoflavones such as genistein may suppress cell mediated immune function. The effect of dietary phytoestrogens on the host response to infection has not been extensively examined. Mice were fed a diet containing soy phytoestrogens and infected with Mycobacterium avium to establish a chronic infection and inflammatory response. As phytoestrogens may act through classical oestrogen receptors (ER), mice deficient in ERalpha signalling and wild type mice were evaluated for a panel of Type 1-associated cytokines (IFNgamma, IL-12 and IL-18) in the spleen. IFNgamma production in the spleen was increased approximately 4-fold in ERalpha-deficient mice fed a casein-based diet over wild type mice fed a casein-based diet (P < 0.05), suggesting a role for ERalpha in suppressing IFNgamma production. IL-18 levels in spleens of wild type mice were decreased compared to ERalpha-deficient mice on a casein diet. Splenic IL-12 and IL-18 levels were not affected in wild type and ERalpha-deficient mice on the phytoestrogen containing diets, with the exception that whole soy increased IL-12 levels in the tissues of ERalpha deficient mice. We conclude that ERalpha and dietary phytoestrogens can influence production of key regulatory cytokines in response to chronic bacterial infection.

In vitro and in vivo regulation of antioxidant response element-dependent gene expression by estrogens.

Understanding estrogen's regulation of phase II detoxification enzymes is important in explaining how estrogen exposure increases the risk of developing certain cancers. Phase II enzymes such as glutathione-S-transferases (GST) and quinone reductase protect against developing chemically induced cancers by metabolizing reactive oxygen species. Phase II enzyme expression is regulated by a cis-acting DNA sequence, the antioxidant response element (ARE). It has previously been reported that several antiestrogens, but not 17beta-estradiol, could regulate ARE-mediated gene transcription. Our goal was to determine whether additional estrogenic compounds could regulate ARE-mediated gene expression both in vitro and in vivo. We discovered that physiological concentrations (10 nm) of 17beta-estradiol repressed GST Ya ARE-dependent gene expression in vitro. Treatment with other endogenous and anti-, xeno-, and phytoestrogens showed that estrogen receptor/ARE signaling is ligand, receptor subtype, and cell type specific. Additionally, GST and quinone reductase activities were significantly lowered in a dose-dependent manner after 17beta-estradiol exposure in the uteri of mice. In conclusion, we have shown that 17beta-estradiol, and other estrogens, down-regulate phase II enzyme activities. We propose estrogen-mediated repression of phase II enzyme activities may increase cellular oxidative DNA damage that ultimately can result in the formation of cancer in some estrogen-responsive tissues.

Re-expression of estrogen receptor alpha in estrogen receptor alpha-negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth.

Estrogen can increase insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) expression, two key components of IGF-I-mediated signaling. The result is sensitization of breast cancer cells to IGF-I and synergistic growth in the presence of estrogen and IGF-I. We hypothesized that loss of estrogen receptor alpha (ERalpha) would result in reduced IGF-mediated signaling and growth. To test this hypothesis, we examined IGF-I effects in MCF-7 breast cancer cell sublines that have been selected for loss of ERalpha (C4 and C4-12 cells are ERalpha-negative) by long-term estrogen withdrawal. C4 and C4-12 cells had reduced IGF-IR and IRS-1 mRNA and protein expression (compared with MCF-7 cells) that was not inducible by estrogen. Furthermore, C4 and C4-12 cells showed reduced IGF-I signaling and failed to show any growth response to either estrogen or IGF-I. To prove that loss of IGF and estrogen-mediated signaling and growth was a consequence of loss of ERalpha, we re-expressed ERalpha in C4-12 cells by stable transfection with HA-tagged ERalpha. Three independent C4-12 ERalpha-HA clones expressed a functional ERalpha that (a) was down-regulated by estrogen, (b) conferred estrogen-induction of cyclin D1 expression, and (c) caused estrogen-mediated increase in the number of cells in S phase. All of the effects were completely blocked by antiestrogens. Interestingly, ERalpha-HA expression in C4-12 cells did not restore estrogen induction of progesterone receptor expression. However, ERalpha-positive C4-12 cells now exhibited estrogen-induction of IGF-IR and IRS-1 levels and responded mitogenically to both estrogen and IGF-I. These data show that ERalpha is a critical requirement for IGF signaling, and to our knowledge this is the first report of functional ERalpha expression that confers estrogen-mediated growth of an ER-negative breast cancer cell line.

Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy.

The formation of mitotic centrosomes is a complex process in which a number of cellular proteins translocate to mitotic poles and play a critical role in the organization of the mitotic apparatus. The 238-kDa nuclear mitotic apparatus protein NuMA is one of the important proteins that plays a significant role in this process. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps of phosphorylations. The role of NuMA in the interphase nucleus is not well known but it is clear that NuMA responds to external signals (such as hormones) that induce cell division, or heat shock that induces apoptosis. In order to determine the function of NuMA it is important to study its localization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen responsive LNCaP prostate cancer cells using immunoelectron microscopy, and on correlation to MPM-2 monoclonal phosphoprotein antibody. These results show that NuMA is present in speckled and punctate form associated with distinct material corresponding to a speckled or punctate immunofluorescence appearance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules while MPM-2 is uniformly distributed in the cytoplasm. During metaphase or anaphase anti-NuMA labeling is associated with spindle fibers. During telophase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MCF-7 and LNCaP cells in the log phase of cell culture growth.

Natural killer cells express estrogen receptor-alpha and estrogen receptor-beta and can respond to estrogen via a non-estrogen receptor-alpha-mediated pathway.

Natural killer (NK) cells play a crucial role in host defense against pathogens and immune surveillance against cancer. Given that estrogens have been reported to suppress NK cell activity, we sought to elucidate the mechanisms by which estrogen mediates this effect. We demonstrate by immunocytochemical staining with estrogen receptor-alpha (ERalpha)- and estrogen receptor-beta (ERbeta)-specific antibodies that both ERalpha and ERbeta are expressed in murine NK cells. We also compared the ability of high doses of 17beta-estradiol ( approximately 800 pg/ml) to regulate NK cell activity in wild-type and estrogen receptor-alpha-deficient (ERalphaKO) mice. 17beta-estradiol elicited a significant decrease in NK cell activity in both wild-type and ERalphaKO mice (P < 0.001). These data suggest that ERbeta or possibly a novel receptor is involved in mediating estrogen action on NK cell activity and raise the potential for therapeutic modulation of NK cell activity with selective estrogen receptor modulators (SERMS).

Hypermethylation of the Wilms' tumor suppressor gene CpG island in human breast carcinomas.

CpG island hypermethylation is known to be associated with transcriptional silencing of tumor suppressor genes in neoplasia. We have previously detected aberrantly methylated sites in the first intron of the Wilms' tumor suppressor (WT1) gene in breast cancer. In the present study, we extended the investigation to a CpG island located in the promoter and first exon regions of WT1. Methylation of this CpG island was found to be extensive in MCF-7 and MDA-MB-231 breast cancer cells, as well as in 25% (five of 20 patients) of primary breast tumors. While levels of the known 3.0-kb WT1 mRNAs were decreased or not detected in these cell lines, the expression could be partially restored following treatment with a demethylation agent, 5-aza-2'-deoxycytidine. Surprisingly, a novel 2.5-kb WT1 transcript was expressed at high levels in both untreated and treated MDA-MB-231 cells. This novel transcript was likely a WT1 variant missing the first exon, and therefore escaped the methylation control present in the normal transcript. Our study implicates the future need to investigate the significance of this aberrant transcript as well as the role of WT1 CpG island hypermethylation in breast neoplasia.

Lithium-stimulated proliferation and alteration of phosphoinositide metabolites in MCF-7 human breast cancer cells.

Lithium, which is used to treat bipolar psychiatric disorders, can stimulate proliferation of a number of cells in tissue culture. Proliferation of MCF-7 human breast cancer cells, which also respond to EGF and estrogens, was stimulated by LiCl (1-5 mM) within the concentration range that is encountered during human therapy with lithium. Stimulation of growth was specific for lithium; rubidium, potassium, and sodium showed no such effect. In the presence of antiestrogen, lithium stimulated the growth of hormone-dependent breast cancer cells MCF-7, ZR-75-1, and T47D but not hormone-independent MDA-MB-231 cells or an estrogen-independent clone of MCF-7 cells. Lithium-stimulated proliferation was limited by cytotoxicity which could be moderated by added potassium chloride (5-20 mM) in the medium. Each of the mitogens lithium, 17 beta-estradiol, and EGF increased the rate of uptake of myo-inositol into MCF-7 cells. Whether normalized to inositol lipids, to protein, or to DNA, steady-state levels of inositol phosphates were elevated by each of the mitogens including lithium, which inhibits the breakdown of inositol phosphates in the phosphoinositide signaling pathway. These data indicate that therapeutic concentrations of lithium can stimulate the proliferation of human breast cancer cells by a mechanism that may involve the phosphoinositide pathway.

Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.

The authors have used morphometric, immunocytochemical, and electron optical techniques to study fibrin deposits associated with villi from 14 normal term placentas, and have examined the response of cultured cellular trophoblast to fibrin matrix in vitro. Morphometric analysis of 3477 villous profiles showed that 5.5% of villi examined had fibrin deposition at sites of syncytial denudation and that fibrin deposition was highly associated with villous epithelial denudation, as evidenced by loss of cytokeratin staining. The perivillous fibrin deposits were strongly immunoreactive for the B beta chain of fibrin II, consistent with local thrombolytic cleavage of fibrinogen to fibrin. Deposits were frequently surfaced by a discontinuous layer of cytokeratin-positive trophoblastic cells that showed type IV basement membrane collagen immunoreactivity at the interface between trophoblast and fibrin. Ultrastructurally, damage to the syncytial trophoblast was apparent at the edge of some deposits, where syncytial denudation was accompanied by a fibrin coating of residual cellular trophoblast and the trophoblastic basal lamina. Other deposits were surfaced by syncytial trophoblast with underlying cellular trophoblast and a new basal lamina external to the basal lamina of the villous core. Cultured cellular trophoblast grown on a fibrin matrix, but not on uncoated plastic, showed morphologic differentiation into a trophoblast layer like that on term villi. The authors suggest that epithelialization of perivillous fibrin deposits is a form of villous repair and that trophoblast-fibrin interactions can modulate trophoblastic differentiation.

Human placental insulin binding in normal and well-controlled diabetic patients.

Previous studies of insulin binding to placentas of both insulin-dependent and untreated gestational diabetic patients have described placentas from diabetics to contain fewer insulin receptors than placentas from nondiabetic gravidas. However, these studies were done using membrane fractions prepared from the placentas and at a time when adequacy of antepartum glycemic control in the diabetic patients was not routinely evaluated by self blood sugar measurement or hemoglobin A1 assay. The current study compares specific 125I-insulin binding in vitro to intact placental villi from 15 normal patients with insulin binding to intact villi obtained from 15 insulin-dependent diabetic mothers whose fasting and postprandial blood sugars and hemoglobin A1 levels were maintained in a range normal for term pregnancy. We demonstrate that insulin binding to intact placental villi is the same in this group of diabetic patients as in the nondiabetic patients.

High glucose levels decrease proliferation of cultured human fetal cells from placenta.

We used the placenta as a source of undifferentiated cells to study the effect high glucose levels can have on human fetal cell proliferation in vitro. Cells were subcultured in a modified minimum essential medium with 10% fetal bovine serum containing either 5.5 mmol/L (100 mg/dl) D-glucose (control), 11 mmol/L (200 mg/dl) D-glucose, or 22 mmol/L (400 mg/dl) D-glucose. Cells grown in mannitol-containing media were used as controls for osmolality. After 3 and 7 days' growth in different media, the labeling index was determined by autoradiographic analysis, and cell numbers were determined with a Coulter counter. The labeling indices for cells grown 3 days in 11 or 22 mmol/L D-glucose were 89% (p less than 0.002) and 84% (p less than 0.001), respectively, of control cells grown in 5.5 mmol/L D-glucose. After 7 days' growth, the labeling indices of cells grown in 11 or 22 mmol/L D-glucose were 84% (p less than 0.002) and 70% (p less than 0.001), respectively, of cells grown in 5.5 mmol/L D-glucose media. There was a significant decrease in the number of cells present at both 3 and 7 days in cultures grown in 22 mmol/L D-glucose compared with control. We conclude that a few day's exposure to high glucose levels can have an effect on proliferation of human placental cells in vitro. We suggest that a glucose effect on proliferation of other cells derived from the products of conception might be one mechanism contributing to abnormal development in some pregnancies of diabetic women.

Morphology and glycoprotein synthesis of uterine glandular epithelium in human basal plate at term: an ultrastructural and autoradiographic study.

This study describes the morphologic features of uterine glandular epithelium in human basal plate at term and identifies this epithelium as an active site of glycoprotein synthesis. Wedge biopsies were obtained from the basal plate at the time of repeat cesarean section from 11 normal pregnant patients at term. Biopsy specimens were either processed immediately for microscopic examination or incubated in vitro with 25 microCi/cc of 3H-galactose or 3H-leucine. Tissues incubated with tritiated compounds (2-hour pulse +/- 3-hour chase in nonradioactive medium) were either processed for light microscopic autoradiographic analysis or extracted for determination of trichloroacetic-acid-precipitable tritiated macromolecules in tissues and medium. Profiles of cuboidal-columnar glandular epithelium were identified in the decidual component of the basal plate region adjacent to spiral arterioles and perpendicular to the inner layers of myometrial muscle. Autoradiographic and biochemical studies identified the glandular epithelium, as well as large decidual cells, to be major sites of incorporation of both 3H-galactose and 3H-leucine and to be prime sources for secretion of tritiated macromolecules that appeared in the medium during in vitro incubation of basal plate tissue. Ultrastructurally, the glandular epithelium was noted to rest on a basal lamina, to have lateral cell membranes with numerous desmosomes, and to exhibit an apical surface with microvilli projecting into a luminal space. Cytologic features of the cells included abundant profiles of rough endoplasmic reticulum, multiple mitochondria with lamellar cristae, a well-developed perinuclear but nonpolarized Golgi apparatus,and nuclei containing predominantly euchromatin.(ABSTRACT TRUNCATED AT 250 WORDS)