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The human endometrium normally undergoes a cyclic proliferation process followed by differentiation under the influence of ovarian steroids and locally produced growth and differentiation factors. Understanding of the molecular mechanisms involved in controlling these processes is of great interest, since imbalances between proliferation- and differentiation-promoting signals can have pathophysiological consequences ranging from infertility to endometrial hyperplasia and tumour formation. The present work reviews aspects of the role played by oncogenes and ovarian steroid receptors in modulating proliferation of endometrial tumour cells. The expression pattern and possible roles of protein kinase C (PKC) subunits are discussed in the context of response-specificity of endometrial tumour cells to tumour-promoting agents such as 12 -O-tetradecanoyl-phorbol acetate (TPA) and possible implications for anti-tumour therapy. [ABSTRACT FROM PUBLISHER]

We have previously shown that the presence of trophoblast cells enhances invasiveness of decidualizing human endometrial stromal cells. The metastasis suppressor CD82, which has antimigratory function in tumor cells, is up-regulated in decidualizing endometrial stromal cells. CEACAM1 is expressed in trophoblast cells at the invasion front in early placenta and is considered proinvasive. Here, we investigate the role of CD82 and CEACAM1 in cocultures of the endometrial stromal cell line T-HESC and AC-1M88 trophoblast cells. In transwell migration assays, chemotaxis of AC-1M88 cells was stimulated by coplated T-HESC in the lower compartment or by the combination of heparin-binding EGF-like growth factor (HB-EGF), interleukin-1 beta (IL-1beta), and leukemia inhibitory factor (LIF), local factors present at the time of implantation. In an implantation model of AC-1M88 trophoblast spheroids on a monolayer of T-HESC, spheroid expansion was enhanced in the presence of HB-EGF/IL-1beta/LIF. Silencing of CEACAM1 in AC-1M88 blunted this response. Chemotactic migration of T-HESC was stimulated by trophoblast secretions or HB-EGF/IL-1beta/LIF. These responses were suppressed by CD82 depletion in T-HESC. Proteome profiling revealed the presence of platelet-derived growth factor (PDGF)-AA in trophoblast supernatant. Chemotaxis of T-HESC toward PDGF-AA was significantly inhibited by CD82 silencing. Neutralization of PDGF-AA in AC-1M88 conditioned media reduced the chemotactic effect on T-HESC. In summary, we demonstrate a mutual stimulation of chemotactic migration between trophoblast and endometrial stromal cells and promigratory roles for the cell surface molecules CEACAM1 and CD82, which may serve to support tissue remodeling at the implantation site.

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site.

Successful pregnancy in humans depends on deep invasion of the maternal decidua by extravillous trophoblast cells (EVTs), a process regulated by autocrine and paracrine signals in the decidual-trophoblast microenvironment. Here we examined whether trophoblast invasion is affected by decidual differentiation of endometrial stromal cells (ESC) and modulated locally by cytokines and growth factors. Trophoblast spheroids were generated from the EVT-derived cell line AC-1M88 and placed onto monolayers of either undifferentiated or decidualized ESC, or directly onto tissue culture surface. Co-cultures were treated with epidermal growth factor (EGF), hepatocyte growth factor, heparin-binding EGF-like growth factor (HB-EGF), interleukin-1β (IL-1β) and leukaemia inhibitory factor (LIF). Expansion of spheroids over 2-3 days was significantly enhanced by a monolayer of undifferentiated ESC compared with tissue culture surface and further increased if ESC had been decidualized. HB-EGF and IL-1β, alone or in combination with LIF, stimulated spheroid expansion but only on undifferentiated ESC. CEACAM1, an adhesion molecule implicated in trophoblast invasion, was up-regulated in AC-1M88 cells by conditioned medium from decidualized ESC, and by HB-EGF, IL-1β and LIF in combination. Treatment of ESC with HB-EGF or IL-1β increased the level of the tetraspanin CD82, a metastasis suppressor found in decidual cells at the implantation site. We suggest that decidualized ESC support trophoblast invasion by paracrine signals that may include HB-EGF, IL-1β and LIF.

Background: Osteopontin (OPN) and its interacting partner CEA-cell adhesion molecule (CEACAM1) mediate similar biological functions and have been expressed in several types of cancer. Here we investigated the prognostic significance of OPN in thyroid tumours and correlated our findings with the expression of CEACAM1., Materials and Methods: 297 human thyroid samples were collected in a tissue microarray and as fresh frozen samples to perform immunohistochemistry and western blotting for OPN and to compare these data with our previously published data on CEACAM1 expression., Results: Nearly all normal samples were negative for OPN. Some thyroid adenomas were weakly OPN positive whereas many carcinomas were strongly positive. In contrast to CEACAM1, which was preferentially expressed in metastatic papillary carcinomas, no associations were found between OPN expression and patient age, gender and tumour size., Conclusion: These results may have future implications for the diagnosis and management of patients with thyroid cancers.

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.

Local release of TGF-beta during times of high bone turnover leads to elevated levels within the bone microenvironment, and we have shown that TGF-beta suppresses osteoclast apoptosis. Therefore, understanding the influences of TGF-beta on bone resorbing osteoclasts is critical to the design of therapies to reduce excess bone loss. Here we investigated the mechanisms by which TGF-beta sustains suppression of osteoclast apoptosis. We found TGF-beta rapidly increased leukemia inhibitory factor (LIF) expression and secretion by phosphorylated mothers against decapentaplegic-dependent and -independent signaling pathways. TGF-beta also induced suppressor of cytokine signaling 3 (SOCS3) expression, which was required for TGF-beta or LIF to promote osteoclast survival by. Blocking LIF or SOCS3 blocked TGF-beta promotion of osteoclast survival, confirming that LIF and SOCS3 expression are necessary for TGF-beta-mediated suppression of osteoclast apoptosis. Investigation of the mechanisms by which LIF promotes osteoclast survival revealed that LIF-induced expression of Bcl-X(L) and repressed Bcl-2 interacting domain expression by activating MAPK kinase, AKT, and nuclear factor-kappaB pathways. Suppression of Janus kinase/signal transducer and activator of transcription signaling further increased Bcl-X(L) expression and enhanced osteoclast survival, supporting that this pathway is not involved in prosurvival effects of TGF-beta and LIF. These data show that TGF-beta coordinately induces LIF and SOCS3 to promote prosurvival signaling. This alters the ratio of prosurvival Bcl2 family member Bcl-X(L) to proapoptotic family member Bcl-2 interacting domain, leading to prolonged osteoclast survival.

Members of the family of carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) belonging to the immunoglobulin (Ig) superfamily are expressed in a variety of normal and malignant human tissues. As components of the cell membrane, these glycoproteins can make contact with adjacent cells. CEACAM1 and CEACAM5 (CEA) express Lewis(x) (Le(x)) structures. As shown by mass spectrometry in conjunction with enzymatic digestion, CEACAM1 contains at least seven Le(x) residues. Fucosyltransferase IX is the main fucosyltransferase responsible for attachment of terminal fucose, the key feature of the Le(x) structure, to CEA and CEACAM1. The Le(x) residues of both, CEACAM1 and CEA, interact with the human Le(x)-binding glycan receptors DC-SIGN and SRCL. Since subpopulations of human macrophages express DC-SIGN or SRCL, Le(x)-carrying CEACAMs may modulate the immune response in normal tissues such as the human placenta or in malignant tumours, for example in colorectal, pancreatic or lung carcinomas., (Copyright (c) 2009 Elsevier GmbH. All rights reserved.)

Background: Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC) decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line., Methods: Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro., Results: St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82). Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages., Conclusion: St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural progesterone. St-T1b cells therefore serve as a useful model for primary ESC.

The aim of this study was to examine the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in pancreatic endocrine tumors (PETs) and to correlate it with clinicopathologic parameters. Sixty-nine PETs were examined for tumor size, necrosis, local peripancreatic invasion and lymphovascular invasion, lymph node, and liver metastasis. The mitotic count, expressed per 10 high-power fields (HPF) and MIB1 index were assessed and tumors were classified according to the World Health Organization classification. A tissue microarray was constructed and stained with an extensive panel of endocrine markers and CEACAM1. Twenty-nine tumors were from males and 40 from females, age range: 23 to 80 years (mean 52.4 y), tumor size ranged from 0.8 to 11 cm (mean 3.5 cm), 8 patients had multiple endocrine neoplasia 1 syndrome, and 1 had von Hippel-Lindau disease. Twenty tumors demonstrated local invasion, 32 had lymphovascular invasion, 16 had lymph node metastasis, and 10 had liver metastasis. CEACAM1 was positive in 47 cases and negative in 22 cases (31.9%). Ninety percent of the CEACAM1-negative cases had a MIB1 index 2% (P=0.02). 86.4% of the CEACAM1-negative PETs had a mitotic count 2/10 HPF. In addition, 80% of tumors >or=2 cm in diameter were CEACAM positive (P<0.05). CEACAM1-positive tumors were more frequently insulin negative (9 of 10 cases) (P=0.005) and vasoactive intestinal peptide-positive PETs were all CEACAM1 immunopositive (7 of 7 cases) (P=0.005). Benign tumors and PETs of uncertain malignant behavior were more frequently CEACAM1-negative and low-grade malignant cases were CEACAM1 positive (27 of 29 cases) (P=0.001). In addition, CEACAM1-positive tumors were statistically correlated with cytokeratin 19-positive tumors (P<0.05). PETs showing CEACAM1 positivity have a statistically significant correlation with several of the pathologic parameters of aggressive behavior and its overexpression is seen in those cases with increased invasiveness.

Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin alphavbeta3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin alphavbeta3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI-h295 cells (>six-fold increase, p < 0.001). Transfection with integrin alphavbeta3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti-integrin beta3 antibody, indicating a functional relationship of OPN and integrin alphavbeta3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan-Meier analysis including 111 patients with primary tumours graded for OPN staining and follow-up data available. In conclusion, our in vitro data indicate that OPN and integrin alphavbeta3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue.

Kangai (KAI)-1 (CD82) is a metastasis suppressor gene, which belongs to the family of tetraspanin proteins. A loss of KAI-1 expression is associated with the advanced stages of many human malignancies. The present study was designed to investigate the expression pattern of KAI-1 in the normal endometrium and uterine tumors and to correlate it with the expression of tumor suppressor protein p53. KAI-1 could be found in the normal endometrium throughout the menstrual cycle. Thirteen of 42 endometrial carcinomas demonstrated moderate KAI-1 expression, but low expression of p53. Twenty-nine of 42 endometrial carcinomas showed reduced or absent KAI-1 expression, which correlated with strong expression of p53 (p < 0.001). There were significant correlations between KAI-1 expression and histological type, e.g., 93% of endometrioid carcinomas displayed a low or moderate immunostaining for KAI-1, whereas nearly all of the serous/clear cell carcinomas were KAI-1 negative (p < 0.001); tumor grading, e.g., 73% of high grade tumors showed no KAI-1 expression (p < 0.001). Most of the investigated uterine sarcomas were negative for KAI-1, whereas they displayed a strong immunostaining for p53. In conclusion, KAI-1 and p53 show inverse expression. The reduced KAI-1 expression may be the result of dysregulated p53 function and could be an important step in the endometrial carcinogenesis.

Fra-2 (Fos-related antigen 2) is a member of the Fos family of AP-1 transcription factors which is often up-regulated in mammary carcinomas. Previous results suggested that it might be involved in the regulation of breast cancer invasion and metastasis. In order to analyze the role of Fra-2 in breast cancer cells, it was silenced in the highly invasive MDA-MB231 cells using RNA interference. On the other hand, stable transfectants of the weakly invasive MCF7 cell line were established in order to analyze the effects of Fra-2 overexpression. In both approaches, cell proliferation was not or only weakly influenced by Fra-2. In contrast, the invasive potential of the cells was increased, and a weaker effect on motility was observed. By cDNA microarray analysis of the MCF7 transfectants followed by validation on a protein level, we identified several Fra-2 target genes which might be involved in cell invasion and migration, i.e., ALCAM and connexin 43. Additionally, mRNA expression levels of various genes which are associated with a more malignant behavior of the tumors in vivo were up- or downregulated, i.e., members of the MAGE family, S100P, TIMP2, IL24 etc. These results show that Fra-2 overexpression is associated with a more aggressive tumor phenotype and is probably involved in breast cancer progression in vivo.

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.

Background: Ovarian cancer represents the malignant tumour of the female genital tract with the worst prognosis, mainly caused by early intraperitoneal spread. Cell-to-cell and cell-to-matrix interactions play a functionally important role in this spread and are both mediated by the cell membrane. Changes in the glycosylation of the cell membrane, as detected by lectin histochemistry, are sometimes associated with a poor prognosis., Patients and Methods: The expression of lectin binding of 164 ovarian cancer patients was analysed and the staining results were correlated with the clinical data of the patients., Results: The univariate and multivariate statistical analysis revealed an independent prognostic significance for Ulex europeus agglutinin-I (UEA-I) binding., Conclusion: These findings indicate that UEA-I binding can serve as a prognostic factor in ovarian cancer.

Objectives: Serum biomarkers for early diagnosis of pancreatic adenocarcinoma are not currently available. We recently observed elevated expression of CEACAM1 in pancreatic adenocarcinomas and sought to determine whether serum CEACAM1 levels were elevated in pancreatic cancer patients., Methods: CEACAM1 messenger RNA levels were measured in pancreatic tissue samples using quantitative reverse transcription-polymerase chain reaction. CEACAM1 was localized by immunohistochemistry in adenocarcinomas and in pancreatic intraductal neoplasia lesions. CEACAM1 serum levels were assessed by a double determinant enzyme-linked immunosorbent assay and compared with serum levels of CA19-9., Results: CEACAM1 had higher expression levels in pancreatic adenocarcinomas compared with noncancerous pancreas (P < 0.0001) and was localized to neoplastic cells (95% (45/47) of adenocarcinomas and 85% (17/20) of pancreatic intraductal neoplasia 3 lesions. CEACAM1 was expressed in the sera of 91% (74/81) of pancreatic cancer patients, 24% (15/61) of normal patients, and 66% (35/53) of patients with chronic pancreatitis, with a sensitivity and specificity superior to CA19-9. The combination of CEACAM1 and CA19-9 had significantly higher diagnostic accuracy than CA19-9., Conclusions: CEACAM1 is expressed in pancreatic adenocarcinoma, and serum levels of CEACAM1 serve as a useful indicator for the presence of pancreatic cancer. Additional validation studies on the use of serum CEACAM1 as a diagnostic marker in pancreatic cancer are warranted.

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.

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.

At the human maternal-fetal interface, the decidua forms a dense matrix that is believed to limit trophoblast invasion. We investigated whether the metastasis suppressor KAI1 (CD82) is expressed at the maternal-fetal interface. Immunohistochemistry showed strong expression of KAI1 in decidual cells, whereas trophoblast cells were negative for KAI1. In luteal phase endometrium, KAI1 was present in decidualizing endometrial stromal cells. We investigated whether KAI1 expression in endometrial stromal cells is regulated by the decidualizing stimuli cAMP and progesterone or by the cytokine interleukin (IL)-1beta. Western blot analysis revealed induction of KAI1 protein by cAMP analog, but not by progesterone, in a delayed fashion. In contrast, IL-1beta rapidly stimulated KAI1 expression at the transcript level and at the protein level. Cultured decidual cells from term placenta expressed a basal level of KAI1 protein that was elevated on cAMP stimulation. Silencing of KAI1 by RNA interference attenuated expression of decorin, a decidual product implicated in limiting trophoblast invasion. This study shows for the first time the expression of KAI1 in decidual cells at the human maternal-fetal interface, where the metastasis suppressor might participate in intercellular communication with trophoblast cells and the control of trophoblast invasion.

Osteopontin (OPN) and CEACAM1 have diverse biological functions in the uterus and placenta throughout the estrous cycle and pregnancy and have been shown to interact with integrin beta3. 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. Recently we showed the expression pattern of OPN in gestational trophoblastic tumors. CEACAM1 is an adhesion molecule of the carcinoembryonic antigen family that we have recently found to be expressed in endometrial cancer and that has been shown to be down-regulated in colorectal, prostate, and breast cancer. In this study, immunohistochemistry and immunofluorescence with specific antibodies were performed on a series of 20 normal endometrial samples, 17 endometrial hyperplasias, and 43 endometrial carcinomas (28 endometrioid, 10 serous, and 5 clear cell carcinomas) to investigate the expression pattern and cell-type specific localization of OPN and to correlate it with the expression of CEACAM1. In addition, Western blot was performed on normal human endometrium and endometrial neoplasia. Strong OPN expression with a consistent cytoplasmic localization in epithelial glandular cells was observed in the normal human endometrium in 80% of the samples of the proliferative and secretory phase (score 8-12). Similar results could be found in endometrial hyperplasias. Strong expression of OPN could be observed in 29 (67.4%) of the 43 analyzed endometrial carcinomas. Of the 43 analyzed tumors, 18 (41.8%) were in the high score (8-12) category with a strong OPN expression level; 11 of 43 (25.5%) showed a moderate score (4-7) category. In endometrioid carcinoma with increasing malignancy grade, increasing areas with low OPN expression level or complete loss of OPN expression could be observed. In contrast, serous tumors showed a strong OPN expression level. Similar results could be found in Western blot analysis. CEACAM1 showed similar results and could be found to be coexpressed with OPN in normal human endometrium and in endometrial neoplasia as we showed using immunofluorescence. In this study, the different expression patterns of OPN in endometrial tumors could additionally support the biological diversity of endometrioid and serous carcinomas together with other markers. We suggest that OPN might play a different role in the pathogenesis of endometrial cancer (possibly as a functional complex with CEACAM1) and could be relevant for invasive growth of such lesions.

Downregulation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), a cell adhesion molecule with tumor suppressing properties has been observed in a high percentage of carcinomas of the endometrium and other malignancies. The mechanisms for the dysregulation and the role of hormones and cytokines on the expression of CEACAM1 in endometrial carcinomas is unknown. We therefore studied the effect of estradiol, medroxyprogesterone acetate (MPA), RU486, gamma-interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 on the expression in the non-expressing endometrial tumor cell lines Hec1B and Skut1B, respectively. No induction of CEACAM1 expression was observed in Hec1B endometrial adenocarcinoma cells in response to hormones and cytokines whereas treatment with TPA and calcium ionophore A23187 resulted in the strong expression of endogenous CEACAM1 on the mRNA and protein levels. In contrast, no induction of CEACAM1 expression was observed in endometrial mixed mesenchymal Skut1B cells. Studies of other members of the CEACAM family revealed that the re-expression in Hec1B carcinoma cells is restricted to CEACAM1 suggesting a cell type-specific and cell type-independent mechanism of CEACAM1 activation via the protein kinase C (PKC) pathway. Induction of CEACAM1 expression was dependent on protein kinase C protein synthesis and luciferase reporter assays with CEACAM1 promoter constructs demonstrated that the re-expression of CEACAM1 is regulated at the transcriptional level. This is the first report demonstrating that activators of PKC are able to specifically induce the expression of CEACAM1 in human carcinoma cells and our findings may provide a basis for the therapeutic inhibition of tumor growth in malignancies in which CEACAM1 is downregulated.

The high-mobility group protein HMGI(Y) is a member of a family of nonhistone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes, and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. Recently we could show that HMGI(Y) is expressed in the normal human placenta, where it is localized in the nuclei of villous cytotrophoblast, in the anchoring villi at the implantation site and in extravillous (intermediate) trophoblast invading the maternal decidua. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. The purpose of this study was to investigate the expression pattern of HMGI(Y) in gestational trophoblastic diseases (GTD), which has not been studied so far. To analyze the expression of HMGI(Y), we performed immunohistochemistry on a total of 29 cases of GTD, including 21 hydatidiform moles and 8 choriocarcinomas. Hydatidiform moles showed a positivity for HMGI(Y) in villous cytotrophoblast and in areas of the trophoblast proliferations on the villous surface; villous syncytiotrophoblast was negative. The choriocarcinomas showed strong immunoreactivity in all cases. The expression pattern of HMGI(Y) in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD and might be potentially useful as an additional diagnostic marker for such lesions.

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.

Abnormalities in the process of trophoblast invasion may result in abnormal placentation. Both the embryonic trophoblast and maternal decidua produce corticotropin-releasing hormone (CRH), which promotes implantation. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is expressed in extravillous trophoblasts (EVTs) of normal human placenta, may also function in tro-phoblast/endometrial interactions. We investigated whether locally produced CRH plays a role in trophoblast invasion, primarily by regulating CEACAM1 expression. We examined cultures of freshly isolated human EVTs, which express CEACAM1, and an EVT-based hybridoma cell line, which is devoid of endogenous CEACAM1. CRH inhibited EVT invasion in Matrigel invasion assays, and this effect was blocked by the CRH receptor type 1 (CRHR1)-specific antagonist antalarmin. Additionally, CRH decreased CEACAM1 expression in EVTs in a dose-dependent manner. After transfection of the hybridoma cell line with a CEACAM1 expression vector, the invasiveness of these cells was strongly enhanced. This effect was inhibited by addition of blocking monoclonal antibody against CEACAM1. Furthermore, blocking of endogenous CEACAM1 in EVTs inhibited the invasive potential of these cells. Taken together these findings suggest that CRH inhibits trophoblast invasion by decreasing the expression of CEACAM1 through CRHR1, an effect that might be involved in the pathophysiology of clinical conditions, such as preeclampsia and placenta accreta.

The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.

Context: The human placenta is a complex tissue and possesses, through its capacity to proliferate and to invade maternal tissue, qualities that are usually found in malignant tumors. Osteopontin (OPN) and CEACAM1 may regulate these processes., Objective: The present study was designed to investigate the expression pattern of OPN in the human placental components and to correlate it with CEACAM1 expression and function in placental cell invasiveness., Design: Immunohistochemistry with an OPN-specific antibody and immunofluorescence were performed on normal placental samples to investigate the expression pattern of OPN and CEACAM1 in the human placenta. Extravillous trophoblast (EVT) hybridoma cells transfected with CEACAM1 and stimulated with OPN were studied using the Matrigel invasion assay., Results: All placentae presented very strong expression of OPN in the EVT at the invasion front, where it colocalized with CEACAM1. In addition, OPN was also present in the villous trophoblast, with strongest expression in the cytotrophoblast of the first trimester. Transfection with CEACAM1 followed by stimulation with OPN resulted in increased invasiveness of EVT hybridoma cells., Conclusion: The present study shows the first systematic analysis of OPN expression pattern in the human placenta showing strong expression in the EVT at the invasion front. Colocalization of OPN with CEACAM1 in the EVT indicates that they might act together to regulate invasiveness at the maternal-fetal interface. Using an in vitro model, we also demonstrated increased cellular invasiveness after OPN treatment. We speculate that OPN and CEACAM1 may act as a functional complex involved in the regulation of placental invasiveness.

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.

The human placenta is an endocrine tissue with a unique capacity for rapid, but tightly controlled, proliferation and invasion. Gestational trophoblastic diseases (GTDs) are placental pathologies with endocrine activity and partially malignant potential and include hydatidiform moles, placental site nodules, and tumors such as placental site trophoblastic tumor and choriocarcinomas. The activating protein-1 (AP-1) family of transcription factors is composed of the cellular homologs of the Jun and Fos oncoproteins, which are immediately involved in cellular proliferation, differentiation, and invasion processes and in the regulation of endocrine genes. The expression pattern of the AP-1 family in the normal human placenta has been recently described, where most of the factors were found in the extravillous (invasive) trophoblast. Their systematic expression in GTD has not been studied thus far. For this reason in this study, we investigated the expression pattern of the AP-1 family in GTDs and compared it with the expression in normal placenta using immunohistochemistry with specific polyclonal antibodies against all members of the AP-1 family (JunB, JunD, c-Jun, c-Fos, FosB, Fra-1, Fra-2). Immunohistochemistry was performed on normal human placentas (positive control) and on 28 cases of GTD including 7 choriocarcinomas and 21 hydatidiform moles. In the normal placenta and in hydatidiform molar samples, most AP-1 factors (especially c-Jun, JunD, and Fra2) were expressed in the intermediate (extravillous) trophoblast. In addition, in molar lesions, strong expression was found in trophoblasts proliferating from the surface of villi. There was only a weak expression of JunB and Fra2 in small fractions of villous cyto- and syncytiotrophoblast nuclei. In choriocarcinomas, there was a strong expression for c-Jun, JunD, Fra1, and Fra2. The specific localization to extravillous trophoblasts and their expression pattern in GTDs indicate that the AP-1 family of transcription factors might be implicated in regulating proliferation and invasion of trophoblasts and play a role in the pathogenesis of GTDs.

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.

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.

Expression of the cell adhesion molecule CEACAM1 in melanomas is an independent factor for the risk of metastasis with a predictive value superior to that of tumor thickness. We have previously shown that CEACAM1 co-localizes at the tumor-stroma interface of invading melanoma masses with integrin beta(3) and that these two adhesion molecules interact via the CEACAM1 cytoplasmic domain. To address the functional consequences of CEACAM1 expression, we investigated invasion and migration of melanocytic and melanoma cells that stably express CEACAM1 using two different in vitro systems. Here, we demonstrate that CEACAM1 expression markedly enhances cell invasion and migration. The enhanced invasion and migration of CEACAM1-transfected cells was dependent on the presence of Tyr-488 within the full-length cytoplasmic CEACAM1 domain. Treatment with anti-CEACAM monoclonal antibodies blocked CEACAM1-enhanced cell invasion and cell migration in a dose-dependent manner. Furthermore, the enhanced invasion and migration of CEACAM1-transfected melanoma cells was blocked by integrin-antagonizing RGD peptides. Expression of integrin beta(3) induces the up-regulation of CEACAM1 in melanocytic MEL6 cells. These results strengthen the view that CEACAM1 and alpha(v)beta(3) integrin are functionally interconnected with respect to the invasive growth of melanomas.

The CCAAT/enhancer-binding protein (C/EBP) family consists of several factors that are important regulators of intracellular processes and hormone action. C/EBP-beta, the most important member of the C/EBP family, was shown recently to be expressed in the normal human placenta where it is localized in villous syncytiotrophoblast and in the extravillous (intermediate) trophoblast but not the villous cytotrophoblast. The purpose of this study was to investigate the expression pattern of C/EBP-beta in gestational trophoblastic disease (GTD) which has not been studied so far. We used immunohistochemistry on a total of 15 cases of GTD including nine complete hydatidiform moles, one placental site nodule (PSN), one placental site trophoblastic tumor (PSTT), and four choriocarcinomas. All our tested specimens showed positivity for C/EBP-beta. The strongest C/EBP-beta expression could be observed in villous syncytiotrophoblast and in the trophoblast proliferations on the villous surface of hydatidiform moles; villous cytotrophoblast was negative. The PSN also showed positive nuclear staining but the expression was not as strong as it was in the hydatidiform moles and the total amount of stained cells was the lowest of all GTD. The PSTT also showed immunoreactivity but with a weaker and more heterogeneous staining than in the choriocarcinomas. The specific expression pattern of C/EBP-beta in GTD indicate that C/EBP-beta could potentially be an additional marker of such lesions.

Members of the Fos family of AP-1 transcription factors (c-Fos, FosB, FosB2, Fra-1 and Fra-2) are able to form dimers with Jun proteins which bind to the regulatory sequences of target genes. As many proteases involved in tumor invasion are AP-1-regulated, we assumed that Fos family members might be important for invasion of mammary carcinomas. Therefore, we performed transient transfections with expression vectors for c-Fos, FosB, FosB2, Fra-1 and Fra-2, followed by matrigel invasion assays. Fra-1 transfection resulted in a 2-4-fold increase of invasive cells in both cell lines. In a less degree, the invasive potential of MDA-MB231 cells was stimulated by Fra-2, whereas MCF7 invasion was enhanced by c-Fos and FosB. By double-labelling immunocytochemistry, PAI-1 up-regulation was observed in cells transfected with c-Fos, Fra-1 and Fra-2 expression vectors, whereas MMP1 and MMP9 expression was not affected. Results of cotransfection with a MMP9 promoter construct and AP-1 expression vectors do not indicate a direct up-regulation of MMP9 expression by Fos proteins except a positive effect of c-Fos in MCF7 cells. In parallel, expression of Fos family members as determined by Western Blot analysis in 75 mammary carcinomas was correlated with MMP1, MMP9, PAI-1 and uPAR protein levels in the tumors. Interestingly, high FosB levels were significantly associated with MMP1 overexpression, whereas expression of c-Fos and phosphorylated Fra-1 correlated with MMP9 protein levels. Strong Fra-2 expression correlated with high levels of MMP9, PAI-1, the uPA/PAI-1 complex and early recurrence. These data indicate that Fos proteins, especially Fra-1, c-Fos and Fra-2, might be involved in invasion of breast cancer cells., (Copyright 2004 Kluwer Academic Publishers)

The human placenta is a tissue with a unique capacity for rapid, but--as opposed to malignant tumours--tightly controlled proliferation and invasion capacity. The members of the activating protein-1 (AP-1) family of transcription factors are key regulators of cellular proliferation, differentiation and invasion processes in many systems and could, thus, play an important role in regulating these processes in the human placenta as well. In the present study, we used immunohistochemistry with specific antibodies against all members of the AP-1 family (c-Jun, JunB, JunD and c-Fos, FosB, Fra-1, Fra-2) to investigate their expression pattern and tissue localization in the human placenta. With the exception of c-Jun, which was expressed in a small fraction of villous cytotrophoblast nuclei and JunD, expressed in some syncytiotrophoblast nuclei, all other members of the AP-1 family were completely absent from villous cyto- and syncytiotrophoblast. Interestingly, most AP-1 factors were expressed in the intermediate (extravillous) trophoblast, with expression being strongest for JunD and Fra2 (100% of nuclei showing strong expression), followed by c-Jun (80% positive nuclei), c-Fos and FosB (50% positive nuclei). This was true for samples of both first trimester and later pregnancy. These data show that, in the human placenta, the AP-1 transcription factors are specifically expressed in the intermediate (extravillous) trophoblast, were they could be implicated in regulating proliferation, differentiation and/or expression of invasion-specific molecules, such as matrix metalloproteinases, which have been shown to be regulated by AP-1 in vitro and are expressed by the invasive trophoblast.

The human trophoblast has the capacity to invade maternal tissue in a controlled fashion and to produce a wide range of hormones. The transcription factors belonging to the CCAAT/enhancer-binding protein (C/EBP) family are regulators of intracellular processes and mediators of hormone action. C/EBP binding sites have been described in the promoters of several placenta-expressed target genes. In the present study, we used immunohistochemistry and Western-blot analysis to investigate the expression pattern of the three most important members of this family, C/EBP-alpha, -beta, and -delta, in the normal human placenta as well as in isolated trophoblast cell populations. We found C/EBP-alpha and C/EBP-beta expression in the villous syncytiotrophophoblast (ST) and the extravillous (intermediate) trophoblast (EVT), but it was absent from the villous cytotrophoblast (CT). Interestingly, expression of C/EBP-beta continued to be very strong up to the third trimester of pregnancy, especially in the ST. C/EBP-delta showed overall lower expression levels, stronger only in the EVT, while CT/ST showed very low/negative expression. These data show for the first time the expression pattern and tissue localization of C/EBP factors in the human placenta, indicating that these factors (especially C/EBP-beta) may play important roles in the regulation of placenta-specific genes and processes.