Invasiveness of human endometrial stromal cells is promoted by decidualization and by trophoblast-derived signals.

BACKGROUND: Extensive invasion of the maternal decidua by extravillous trophoblast is considered of critical importance for implantation and placentation in humans, the decidua being viewed as a passively invaded tissue. In this study, we examined whether decidual cells might contribute to the highly dynamic processes at the fetal-maternal interface by active movement. METHODS: Primary endometrial stromal cells (ESCs) or the telomerase-immortalized ESC line, St-T1b, was induced to decidualize or was left undifferentiated. The AC-1M88 cell line served as a model for extravillous trophoblast cells. Motility of ESCs and trophoblast cells was monitored in transwell invasion and migration assays under co-culture conditions. Secretion of matrix metalloproteinases (MMPs) was assessed by gelatin zymography. RESULTS: AC-1M88 cell invasiveness was unaffected by the presence of ESCs, irrespective of their decidualization status. Surprisingly, decidualized ESCs were significantly more invasive than undifferentiated cells, and this invasive activity was strongly enhanced when cells were cultured in direct contact with AC-1M88 cells. Conditioned medium from AC-1M88 cells also stimulated migration and invasion of ESCs. Secretion of MMP-2 and -9 by ESCs was increased upon decidualization. CONCLUSIONS: Enhanced motility and invasive capacity of decidualized ESCs in the presence of trophoblastic cells lead us to hypothesize a major contribution of the decidua in encapsulating the early conceptus and supporting subsequent trophoblast invasion. Our findings thus suggest a far more active role of the decidua in the implantation process than hitherto recognized.

Mechanism and functional consequences of loss of FOXO1 expression in endometrioid endometrial cancer cells.

The forkhead transcription factor FOXO1, a downstream target of phosphatidylinositol-3-kinase/Akt signalling pathway, regulates cyclic differentiation and apoptosis in normal endometrium, but its role in endometrial carcinogenesis is unknown. Screening of endometrial cancer cell lines demonstrated that FOXO1 is expressed in HEC-1B cells, but not in Ishikawa cells, which in turn highly express the FOXO1 targeting E3-ubiquitin ligase Skp2. FOXO1 transcript levels were also lower in Ishikawa cells and treatment with the proteasomal inhibitor was insufficient to restore expression. Lack of FOXO1 expression in Ishikawa cells was not accounted for by differential promoter methylation or activity, but correlated with increased messenger RNA (mRNA) turnover. Comparative analysis demonstrated that HEC-1B cells proliferate slower, but are more resistant to paclitaxel-mediated cell death than Ishikawa cells, which were partially reversed upon silencing of FOXO1 in HEC-1B cells or its re-expression in Ishikawa cells. We further show that FOXO1 is required for the expression of the growth arrest- and DNA-damage-inducible gene GADD45alpha. Analysis of biopsy samples demonstrated a marked loss of FOXO1 and GADD45alpha mRNA and protein expression in endometrioid endometrial cancer compared to normal endometrium. Together, these observations suggest that loss of FOXO1 perturbs endometrial homeostasis, promotes uncontrolled cell proliferation and increases susceptibility to genotoxic insults.

Expression of urocortin in the extravillous human trophoblast at the implantation site.

Urocortin (UCN) is a 40 amino acid peptide which is closely related to corticotropin-releasing hormone and binds with high affinity to both CRH type 1 and type 2 receptors. UCN is expressed in human reproductive tissues including endometrium, ovary, and placenta. This study was designed to investigate the cellular localization of UCN at the implantation site of the human blastocyst, as well as the regulation of the UCN promoter by two major intracellular signaling pathways, the cAMP/PKA and diacylglycerol/PKC pathways, in cells of placental origin. For this reason, immunohistochemistry was performed on tissue sections from paraffin-embedded human first trimester placentas and freshly isolated human invasive extravillous trophoblast cells (EVT) were analyzed for UCN expression using RT-PCR and immunofluorescence. Finally, UCN promoter activity was analyzed in the JEG3 human choriocarcinoma cell line. Immunohistochemistry revealed expression of UCN in the cytotrophoblast, the EVT and decidual cells. Both UCN mRNA and peptide were detectable in freshly isolated EVT. Finally, a human UCN promoter luciferase reporter construct transfected into JEG3 cells was significantly inducible by phorbol ester plus ionomycin, but not by phorbol ester alone or by forskolin. Collectively, the present study reports the expression of UCN in EVT and the activation of the UCN gene promoter by the diacylglycerol/PKC pathway. The functional significance of urocortin for the physiology of EVT requires further investigation.

CEACAM1 impedes thyroid cancer growth but promotes invasiveness: a putative mechanism for early metastases.

CEACAM1, also known as biliary glycoprotein (BGP), CD66a, pp120 and C-CAM1, is a member of the CEA immunoglobulin superfamily. CEACAM1 is a putative tumor suppressor based on diminished expression in some solid neoplasms such as colorectal carcinoma. However, CEACAM1 is overexpressed in some tumors such as non-small cell lung cancer. To clarify the mechanism of action of this cell adhesion molecule, we studied thyroid carcinoma that has a spectrum of morphologies and variable behavior allowing separation of proliferation from invasion and metastasis. CEACAM1 is expressed in thyroid carcinoma cell lines derived from tumors that exhibit aggressive behavior. Introduction of CEACAM1 into endogenously deficient WRO cells resulted in reduced cell cycle progression associated with p21 upregulation and diminished Rb phosphorylation. Forced CEACAM1 expression enhanced cell-matrix adhesion and migration and promoted tumor invasiveness. Conversely, small interfering RNA (siRNA)-mediated downregulation of CEACAM1 expression in MRO cells accelerated cell cycle progression and significantly enhanced tumor size in xenografted mice. CEACAM1 is not appreciably expressed in normal thyroid tissue or benign thyroid tumors. In a human thyroid tissue array, CEACAM1 reactivity was associated with metastatic spread but not with increased tumor size. These findings identify CEACAM1 as a unique mediator that restricts tumor growth whereas increasing metastatic potential. Our data highlight a complex repertoire of actions providing a putative mechanism underlying the spectrum of biologic behaviors associated with thyroid cancer.

Expression of the glucocorticoid receptor in the human adrenal cortex.

Glucocorticoids produced in the adrenal cortex act by binding to a specific intracellular protein, the glucocorticoid receptor (GR), which then modulates gene transcription in target tissues. Whether the adrenal cortex itself is a glucocorticoid target tissue has not been analyzed as yet. Since the presence of GR would be a prerequisite for such "intracortical" glucocorticoid action, this study was designed to analyze GR expression in the normal human adrenal gland using RT-PCR, Western blot, and immunohistochemistry. RT-PCR revealed the presence of GR mRNA in adrenal cortex as well as in NCIh295 cells. These results were confirmed at the protein level by Western blot employing a specific anti-human GR antibody. Immunohistochemically, weak GR staining was observed in the adrenal medulla. In contrast, GR was strongly expressed in the adrenal cortex with the zona reticularis showing the most intense staining. Transfection of a GR-responsive luciferase reporter gene into NCIh295 cells resulted in dexamethasone-dependent induction of luciferase activity, indicating that GR is functional in this tissue. In this study, we show for the first time that GR is expressed in the human adrenal cortex. Its preferential expression in the zona reticularis may indicate a functional role in the regulation of adrenal androgen biosynthesis.

Expression and prognostic relevance of activated extracellular-regulated kinases (ERK1/2) in breast cancer.

Extracellular-regulated kinases (ERK1, ERK2) play important roles in the malignant behaviour of breast cancer cells in vitro. In our present study, 148 clinical breast cancer samples (120 cases with follow-up data) were studied for the expression of ERK1, ERK2 and their phosphorylated forms p-ERK1 and p-ERK2 by immunoblotting, and p-ERK1/2 expression in corresponding paraffin sections was analysed by immunohistochemistry. The results were correlated with established clinical and histological prognostic parameters, follow-up data and expression of seven cell-cycle regulatory proteins as well as MMP1, MMP9, PAI-1 and AP-1 transcription factors, which had been analysed before. High p-ERK1 expression as determined by immunoblots correlated significantly with a low frequency of recurrences and infrequent fatal outcome (P = 0.007 and 0.008) and was an independent indicator of long relapse-free and overall survival in multivariate analysis. By immunohistochemistry, strong p-ERK staining in tumour cells was associated with early stages (P = 0.020), negative nodal status (P = 0.003) and long recurrence-free survival (P = 0.017). In contrast, expression of the unphosphorylated kinases ERK1 and ERK2 was not associated with clinical and histological prognostic parameters, except a positive correlation with oestrogen receptor status. Comparison with the expression of formerly analysed cell-cycle- and invasion-associated proteins corroborates our conclusion that activation of ERK1 and ERK2 is not associated with enhanced proliferation and invasion of mammary carcinomas.

Osteopontin expression in gestational trophoblastic diseases: correlation with expression of the adhesion molecule, CEACAM1.

The human placenta is a complex tissue with multiple endocrine and nutritional functions and a unique capacity for rapid proliferation but tightly controlled invasion, differentiating it from malignant tumors. Osteopontin (OPN) is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. OPN also could be implicated in regulating implantation and placentation by promoting cellular migration and invasion in a placenta-specific fashion. We could demonstrate the expression pattern of OPN in the normal human placenta in which it is localized in the extravillous (intermediate) trophoblast and the villous cytotrophoblast. CEACAM1 is an adhesion molecule, which we have recently found to be expressed at the maternal-fetal interface of the normal placenta with a localization to the extravillous (invasive) trophoblast and in gestational trophoblastic disease (GTD) and also to be potentially implicated in trophoblast invasion and tumorigenesis. Both OPN and CEACAM1 have been shown to interact with integrin beta3. The purpose of this study was to investigate the expression pattern of OPN in GTD and to correlate it with the expression of CEACAM1. To analyze the expression of OPN, we performed immunohistochemistry on a total of 27 cases of GTD, including 21 hydatidiform moles and 6 choriocarcinomas, which had previously been characterized with respect to their CEACAM1 expression. Hydatidiform moles showed a positivity for OPN in villous cytotrophoblast and in the trophoblast proliferations on the villous surface. The strongest OPN expression could be observed in the choriocarcinomas with a heterogenous OPN expression pattern. CEACAM1 had shown similar results and was found to be expressed in choriocarcinoma. The expression pattern of osteopontin in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD (possibly as a functional complex with CEACAM1 and integrin beta3) and might be useful as an additional diagnostic marker for such lesions.

Vitamin B6 modulates glucocorticoid-dependent gene transcription in a promoter- and cell type-specific manner.

Due to their immunosuppressive effects, glucocorticoids (GC) are widely used in the treatment of inflammatory and autoimmune states. However, long-term GC treatment is associated with severe side effects. To increase the ratio of wanted and unwanted GC effects, is, therefore, a desirable goal, which could be achieved by either developing new "dissociating" GC or by combining conventional GC therapy with substances that selectively interfere with glucocorticoid receptor (GR) function. Vitamin B6 was previously shown to inhibit GR transactivation in non-immune cells. In the present study, we tested whether vitamin B6 would also interfere with GR function in immune cells and/or with transrepression in non-immune cells. Normal human lymphocytes and Jurkat T lymphoma cells were transfected with luciferase reporter constructs under the control of the interleukin-2 (IL-2) and the leukemia inhibitory factor (LIF) promoter, respectively. Cells were stimulated with phorbol ester, ionomycin, and different concentrations of dexamethasone, either in the absence (a vitamin B6-free medium was especially prepared for this study) or presence of vitamin B6. Both promoters were strongly induced in response to phorbol ester and ionomycin. Dexamethasone inhibited this effect in a dose-dependent manner both in the presence and absence of vitamin B6. Similar results were obtained at the protein level (IL-2- and LIF-specific ELISAs). Induction of a glucocorticoid response element (GRE)-driven promoter construct by dexamethasone in lymphoid cells was only marginally reduced by vitamin B6. In contrast, GR-mediated transactivation was strongly inhibited by vitamin B6 in HeLa cells, while GR-mediated transrepression of a matrix metalloproteinase 9 (MMP9) promoter construct was not affected. Our data indicate that vitamin B6 does not interfere with GC action in immune cells (wanted GC effects) while selectively inhibiting GR-dependent transactivation in non-immune cells (unwanted GC effects). Combination of GC treatment with supraphysiological doses of vitamin B6 may, thus, reduce the side effects of this type of immunosuppressive therapy, provided that the observed effects can be reproduced at subtoxic vitamin B6 concentrations in vivo.

Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene.

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

The glucocorticoid receptor is specifically expressed in the stromal compartment of the human endometrium.

The human endometrium is a classical target tissue for steroid hormones. While the expression pattern and functional roles of both the estrogen receptor (ER) and the progesterone receptor (PR) are well defined, expression of the glucocorticoid receptor (GR) in this tissue has not been described so far. In the present study, we used immunohistochemistry to analyze the expression of GR in the normal human endometrium throughout the menstrual cycle. The expression of GR was compared to that of ER and PR, which were analyzed in parallel. We show that GR is expressed in the human endometrium with a pattern that markedly differs from the expression patterns of ER and PR. ER and PR are expressed in the nuclei of endometrial glands, whereas GR is completely absent from these structures. However, GR is strongly expressed in the stromal compartment of the endometrium throughout the cycle. Both stromal fibroblasts and lymphocytes are GR-positive. In addition GR expression is also observed in the endothelium of small endometrial blood vessels, which are ER- and PR-negative. Western blot analysis performed on endometrial tumor cell lines of glandular (HEC-1B) and mesodermal (SKUT-1B) origin, respectively, showed GR expression only in the latter. In summary, we demonstrate that GR is expressed in fibroblasts, lymphocytes and endothelial cells of the human endometrial stroma, while it is absent from the glandular compartment. The specific expression pattern of GR within the human endometrium points to a possible functional role of glucocorticoids in the process of decidualization which occurs primarily in the stromal compartment.

Expression of cell-cycle regulatory proteins in endometrial carcinomas: correlations with hormone receptor status and clinicopathologic parameters.

PURPOSE: The normal human endometrium is characterized by hormone-dependent variations in the levels of cell-cycle regulatory proteins during the menstrual cycle. As this tightly controlled system is disturbed in endometrial carcinomas, we analyzed which cell-cycle regulators are involved in endometrial carcinogenesis. METHODS: We performed Western blot analysis of five cell-cycle stimulating (cyclins D1, E, B1, cdk2, cdk4) and three cell-cycle inhibiting (p16(INK4a), p21(WAF1), Rb) proteins in 41 endometrial carcinoma specimens. In addition, expression of the estrogen and progesterone receptors (ER, PR), Ki67, and, in selected cases, p16, cyclin E, and cyclin B1 was studied by immunohistochemistry. RESULTS: We found upregulation of all analyzed cell-cycle regulators in most tumors compared to normal endometrial tissue samples. Overexpression of cyclin E, cyclin B1, and p21 was associated with a less differentiated phenotype. In addition, high levels of cyclin E, cdk2, and cdk4 correlated with weak/absent ER expression, and p16 and p21 overexpression was significantly associated with low PR immunoreactivity. Cyclin B1 expression correlated with cyclin E, cdk2, cdk4, p21, Rb, and Ki67, and cyclin E expression with cyclin D1 and Rb. CONCLUSIONS: We conclude that cyclin E and cyclin B1 might be the major cell-cycle regulators involved in proliferation and reduced differentiation of endometrial carcinomas. In addition, p16, p21, and Rb appear to be uncoupled from their normal cell-cycle inhibiting function in many endometrial carcinomas.

Expression pattern of the AP-1 family in endometrial cancer: correlations with cell cycle regulators.

PURPOSE: To study the expression pattern and the role of the AP-1 (activating protein-1) family of transcription factors in endometrial carcinogenesis. METHODS: We performed Western blot experiments with specific antibodies for each of the AP-1 proteins (c-jun, junB, junD, c-fos, fosB, fra-1, fra-2) with 41 endometrial carcinomas. Expression levels of the AP-1 factors were correlated with clinico-pathologic tumor parameters, steroid receptor status, Ki-67 expression and the expression levels of eight cell cycle regulatory proteins (cyclin D1, cyclin E, cyclin B1, cdk2, cdk4, p16, p21, and Rb). RESULTS: Of the seven AP-1 factors, three (c-fos, fra2, and junB) clearly showed higher expression levels in tumors when compared to matched normal endometrial samples. These factors also correlated significantly with cell cycle promoters (c-fos with cyclin E, cyclin B1, cdk2, and cdk4; fra-2 with cyclin B1; and junB with cyclin D1). Furthermore, high c-fos expression correlated with low ER and PR immunoreactivity and high grading (G3). On the other hand, correlations with classic cell cycle inhibitors (Rb, p16, p21) have also been observed for all AP-1 factors except c-jun and junD. CONCLUSIONS: Our results indicate that the AP-1 family of transcription factors is probably implicated in the regulation of cell cycle progression and control in endometrial carcinomas. In particular, c-fos might be an additional negative prognostic factor and/or implicated in tumor progression in endometrial cancer.

cis Interaction of the cell adhesion molecule CEACAM1 with integrin beta(3).

CEACAM1 is a cell adhesion molecule that has been implicated in a number of physiological processes (eg, tumor suppressor in epithelial tissues, potent angiogenic factor in microvessel formation, microbial receptor in human granulocytes and epithelial cells). The mechanism of CEACAM1 action is still largely unresolved but recent findings demonstrated that the cytoplasmic CEACAM1 domain is linked indirectly to the actin-based cytoskeleton. We have isolated integrin beta(3) as an associated protein using CEACAM1 tail affinity purification. This association depends on phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain. Confocal laser scanning microscopy confirmed in vivo colocalization of both molecules in human granulocytes and epithelial cells. Furthermore, the concentrated colocalization at the tumor-stroma interface of invading melanoma masses suggests a functional role of CEACAM1-integrin beta(3) interaction in melanoma invasion. Moreover, colocalization of the two adhesion molecules is also found at the apical surface of glandular cells of pregnancy endometrium. Colocalization of CEACAM1 and integrin beta(3) at the transitional zone from proliferative to invasive extravillous trophoblast of the maternal-fetal interface supports a role for CEACAM1/integrin beta(3) complexes in cell invasion.

Expression of Rb2/p130 in breast and endometrial cancer: correlations with hormone receptor status.

Rb2/p130 is a member of the retinoblastoma family of proteins, consisting of Rb, Rb2 and p107, which are important negative regulators of cell cycle progression and differentiation. While Rb2 downregulation was observed in several malignant tumours including endometrial cancer, the role of p130 in breast carcinomas is still unknown. We investigated Rb2 protein expression in tumour tissue from 68 mammary and 41 endometrial carcinomas, 4 mammary cell lines, and normal tissue samples. Therefore, we performed Western blot experiments for Rb2, Rb, and the oestrogen and progesterone receptors (ER, PR-A, PR-B). Weak or absent Rb2 expression was more often found in endometrial (59%) than in mammary carcinomas (24%). We found significant positive correlations of Rb2 expression with Rb, ER, and PR-B in breast cancer samples, and of Rb2 with Rb, PR-A, PR-B, and younger age in endometrial carcinomas. No significant associations with histological grading, stage, nodal involvement, or Ki67 staining were detected. Rb2 mRNA expression was studied by semi-quantitative RT-PCR in 56 endometrial or mammary tissue samples and correlated significantly with Western blot results. Our results indicate that loss of Rb2 expression, mostly by transcriptional down-regulation, may be associated with the development and dedifferentiation of most endometrial and a subset of mammary carcinomas.

Overexpression of the p16 cell cycle inhibitor in breast cancer is associated with a more malignant phenotype.

In order to study the role of the p16INK4A(MTS1/CDKN2a) tumor suppressor in breast cancer, we analyzed p16 protein expression in 60 breast cancer samples which were also analyzed for expression of Rb, Ki67, HER2/neu, and estrogen and progesterone receptors (ER, PR). P16 expression was investigated by two methods: western blotting (WB) followed by densitometry, and immunohistochemistry (IHC). The Rb status was studied by western blotting, and expression of Ki67, HER2/neu, ER, and PR was analyzed immunohistochemically. P16-negative results were found in 18% of the carcinomas by WB, but in only one case by IHC and were not associated with established prognostic parameters. In contrast, p16 overexpression which was detected by WB and IHC in 15% and 25% of the tumors, respectively, was significantly associated with unfavorable prognostic indicators. High p16 expression as detected by both methods correlated significantly with high grading and a negative estrogen receptor status. In addition, a significant association of p16 staining with inverse progesterone receptor status and high Ki67 expression was found with IHC. No correlation of p16 expression with clinical stage, HER2/neu immunostaining, Rb expression or Rb phosphorylation was found. Comparison of western blot results and immunohistochemistry suggests that both nuclear and cytoplasmic immunoreactivity in tumor cells is specific and due to p16 expression. We conclude that high p16 reactivity (both nuclear andcytoplasmic) is indicative of a more undifferentiated, malignant phenotype in mammary carcinomas.

Expression pattern of the adhesion molecule CEACAM1 (C-CAM, CD66a, BGP) in gestational trophoblastic lesions.

CEACAM1 (CD66a, BGP, C-CAM) is an adhesion molecule of the carcinoembryonic antigen (CEA) family which has been shown to be normally expressed at the apical pole of epithelial cells and to show a dysregulated expression pattern in tumors derived from the latter. The purpose of the present study was to investigate the expression pattern of CEACAM1 in gestational trophoblastic lesions and to compare this expression with the one observed in the normal trophoblast. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody which specifically recognizes CEACAM1 and does not interact with other members of the CEA family. Immunohistochemistry was performed on a total of 20 cases of gestational trophoblastic lesions including complete hydatidiform moles, one placental site trophoblastic nodule (PSN), one placental site trophoblastic tumor (PSTT), and three choriocarcinomas. Immunostaining for cytokeratin, hPL, hCG, and Ki-67 was also performed. Normal placental samples served as a control. CEACAM1 was absent from villous cyto- and syncytiotrophoblast in both normal placenta and hydatidiform molar samples. It was present in the benign extravillous trophoblast, with stronger expression in the proximal extravillous trophoblast of anchoring villi, but was also observed in interstitial and endovascular intermediate trophoblast and chorionic intermediate-like trophoblast. Partial expression was observed in the trophoblast proliferating from the surface of molar villi. In choriocarcinomas, areas of weak expression could be observed along with large areas without CEACAM1 expression. In the PSN and especially in the PSTT, CEACAM1 expression was stronger and more diffuse. The specific localization to extravillous trophoblast and its expression pattern in gestational trophoblastic lesions indicate that CEACAM1 can potentially be a helpful additional diagnostic marker in the differential diagnosis of such lesions.

Expression and tissue localization of soluble guanylyl cyclase in the human placenta using novel antibodies directed against the alpha(2) subunit.

The cytoplasmic or soluble forms of guanylyl cyclase (sGC) are heme-containing heterodimeric enzymes that are regulated by nitric oxide (NO) and carbon monoxide (CO). These gaseous messenger molecules are produced in the human placenta and are potential regulators of vasodilation and trophoblast invasion. The alpha(2)-subunit of sGC has only recently been shown to naturally occur in placental extracts. In the present study, two novel antibodies directed against different epitopes of the alpha(2) subunit, were generated. Western Blot analysis confirmed the presence of a 82 kDa protein, identical with alpha(2) protein overexpressed in Sf9 cells. According to RNase protection analysis the alternatively spliced alpha(2i) variant was absent from human placenta. Immunohistochemical analysis showed the presence of alpha(2) protein in syncytiotrophoblast and villous and umbilical blood vessels, which are known sites of NO production. Strong expression was observed in the extravillous (intermediate) trophoblast, where the expression of CO-generating hemeoxygenases has recently been documented. Localization of alpha(2) subunit expression suggests a role for sGC in mediating the actions of both NO and CO. The novel antibodies characterized in the present study will be powerful tools to further elucidate the role of the NO/CO/cGMP signaling pathways in pathologic states such as preeclampsia and intrauterine growth retardation.

PTEN expression in breast and endometrial cancer: correlations with steroid hormone receptor status.

OBJECTIVE: The PTEN (MMAC1/TEP1) tumor suppressor gene is frequently mutated and homozygously deleted in human neoplasms, but there is only sparse information about PTEN protein expression in hormone-dependent female tumors. Therefore, we investigated PTEN expression in 68 breast and 43 endometrial carcinomas. METHODS: For PTEN protein detection, we used Western blot analysis followed by densitometry and compared these data with clinicopathologic parameters, the estrogen receptor (ER) and progesterone receptor (PR) status, HER2/neu and the proliferation marker Ki67. RESULTS: We were able to show significantly decreased PTEN protein expression in endometrial carcinomas compared with normal endometrial tissue samples, especially in the endometrioid histological subtype. In contrast, PTEN downregulation was found more rarely in breast cancer. Lower PTEN expression in breast cancer correlated significantly with high ER immunoreactivity (p = 0.008) and was weakly associated with PR expression (p = 0.055) and low histological grading (p = 0.081). No correlation with any of these parameters was observed in endometrial tumors. In both tumor types, no association of PTEN expression with any other analyzed parameter was found. CONCLUSIONS: These results suggest that PTEN expression plays different roles in the pathogenesis of endometrial carcinomas and breast cancer. In mammary carcinomas, loss of PTEN expression is mainly found in more differentiated tumors and is probably not a major event in carcinogenesis.

Biphasic effect of medroxyprogesterone-acetate (MPA) treatment on proliferation and cyclin D1 gene transcription in T47D breast cancer cells.

While progesterone is a known differentiation-inducing factor in the human endometrium, for the breast epithelium both proliferation-inducing and -inhibiting effects have been described. Cyclin D1, which is required for cell cycle progression in G1 and has been shown to play an important role in the pathogenesis of breast cancer has been implicated as a possible mediator of such effects. In the present study we thus investigated the effects of the progestin agonist MPA (medroxy-progesterone acetate) on proliferation of T47D breast cancer cells. In parallel experiments, the regulation of the human cyclin D1 promoter as well as cyclin D1 protein levels under the influence of MPA were studied. Our results show an increase of proliferative activity in T47D cells after 24 and 48 h of MPA treatment followed by inhibition of proliferation after 72 h. In Western blot analysis an increased expression level of cyclin D1 protein can be observed after 24h of MPA stimulation, while at 72h the protein levels are barely detectable. Transient transfection experiments with a luciferase reporter plasmid containing the human cyclin D1 promoter showed an induction of the promoter after 24 and 36h of MPA treatment followed by a reduction in promoter activity. In conclusion, our results confirm the existence of a biphasic response of T47D cell proliferation in response to MPA treatment, consisting of stimulation of proliferation followed by inhibition, and further implicate cyclin D1 as a mediator of these effects, since the cyclin D1 promoter shows a similar biphasic response in this context.