PRV-1 mRNA expression discriminates two types of essential thrombocythemia.

Essential thrombocythemia (ET) is a heterogeneous disorder. For example, the growth of erythropoietin-independent erythroid colonies, termed "endogenous erythroid colonies (EECs)", has previously been observed in only 50% of ET patients. We have recently described the overexpression of a hematopoietic receptor, PRV-1 (polycythemia rubra vera-1), in patients with polycythemia vera (PV). Here, we compare PRV-1 expression and EEC formation in a cohort of 30 patients with ET; 50% of the ET patients in our cohort displayed EEC growth. Likewise, 50% of the ET patients overexpressed PRV-1. Remarkably, only the 15 ET patients displaying EEC growth showed elevated PRV-1 expression, while the 15 EEC-negative ET patients expressed normal PRV-1 levels. It has previously been reported that EEC-positive ET patients develop PV during long-term follow-up. Here, we show that 40% of the PRV-1-positive patients develop symptoms of PV during the course of their disease. In contrast, none of the 15 PRV-1-negative patients displayed such symptoms (p=0.017). Moreover, PRV-1-positive patients had a significantly higher number of thromboembolic or microcirculatory events (p=0.003). We propose that PRV-1-positive ET comprise a pathophysiologically distinct subgroup of patients, one that is at risk for the development of complications and for the emergence of PV.

Cysteine 38 in p65/NF-kappaB plays a crucial role in DNA binding inhibition by sesquiterpene lactones.

Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-kappaB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-kappaB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IkappaB-alpha degradation. Here we report the construction of p65/NF-kappaB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,120) --> Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-kappaB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Although a slight inhibition of IkappaB degradation was detected for all SLs, the amount of remaining IkappaB was too low to explain the observed NF-kappaB inhibition.

Expression of l-3-phosphoserine phosphatase is regulated by reconstituted basement membrane.

Reconstituted basement membrane (Matrigel) promotes differentiation of endometrial adenocarcinoma cells in vitro. However, little is known about the molecular basis of these in vitro differentiation processes. Using differential display RT-PCR to search for potential molecular markers we screened for genes which respond to contact to basement membrane by alteration of expression levels. Here we report that the cDNA MT32 represents an mRNA with a time dependent biphasic response pattern to contact to basement membrane. Characterizing MT32 revealed that the sequence of MT32 is identical to l-3-phosphoserine phosphatase. PCR analysis of l-3-phosphoserine phosphatase expression surprisingly revealed at least three variants of this enzyme. In summary, and in view of the literature, l-3-phosphoserine phosphatase and potential variants or family members represent molecular markers to study regulation of gene expression by components of the extracellular matrix. In conclusion, l-3-phosphoserine phosphatase(s) may be important in endometrial carcinogenesis since this enzyme synthesizes important metabolic intermediates which serve both as building blocks for peptide synthesis and for signal transducing molecules.

Relative binding affinity does not predict biological response to xenoestrogens in rat endometrial adenocarcinoma cells.

The possible adverse effects of the so-called environmental estrogens have raised considerable concern. Developmental, endocrine and reproductive disorders in wildlife animals have been linked to high exposure to persistent environmental chemicals with estrogen-like activity (xenoestrogens); yet, the potential impact of environmental estrogens on human health is currently under debate also due to lack of data. A battery of in vitro assays exist for identifying compounds with estrogenic activity, but only a few models are available to assess estrogenic potency in a multiparametric analysis. We have recently established the endometrial adenocarcinoma cell line RUCA-I; it enables us to compare estrogenic effects both in vitro and in vivo as these cells are estrogen responsive in vitro and grow estrogen sensitive tumors if inoculated in syngeneic animals in vivo. Here we report in vitro data concerning (a) the relative binding affinity of the selected synthetic chemicals Bisphenol A, nonylphenol, p-tert-octylphenol, and o,p-DDT to the estrogen receptor of RUCA-I cells and (b) the relative potency of these compounds in inducing increased production of complement C3, an endogenous estrogen-responsive gene. Competitive Scatchard analysis revealed that xenoestrogens bound with an at least 1000-fold lower affinity to the estrogen receptor of RUCA-I cells than estradiol itself, thereby exhibiting the following affinity ranking, estradiol>>>nonylphenol>bisphenol A approximately p-tert-octylphenol>o,p-DDT. Despite these low binding affinities, bisphenol A, nonylphenol and p-tert-octylphenol increased production of complement C3 in a dose dependent manner. Compared with estradiol, only 100-fold higher concentrations were needed for all the compounds to achieve similar levels of induction, except o,p-DDT which was by far less potent. Northern blot analyses demonstrated that the increased production of complement C3 was mediated by an increased transcription. In summary, cultured RUCA-I cells represent a valuable endometrial derived model system to assess the relative potencies and the molecular mode of action of environmental estrogens in vitro. Our results further show that no intimate correlation exists between the relative binding affinity and the biological response of these compounds. Therefore, data obtained from single-parametric analyses may result in misleading conclusions. On the other hand, the presented in vitro data will provide us with tools to study the activity of xenoestrogens in vivo and thus carry risk assessment one step further.

Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenicity profiles and uterotropic activity.

The function of the uterus is regulated by female sex steroids and it is, therefore, used as the classical target organ to detect estrogenic action. Uterine response to estrogens involves the activation of a large pattern of estrogen-sensitive genes. This fact offers the opportunity to analyze the estrogenic activity of xeno- and phytoestrogens, and the mechanisms of their molecular action by a correlation of the uterotropic activity and their ability to modulate the expression of estrogen-sensitive genes. We have analyzed the expression of androgen receptor (AR), progesterone receptor (PR), estrogen receptor (ER), clusterin (CLU), complement C3 (C3), and GAPDH mRNA in the rat uterus following oral administration of ethinylestradiol (EE), bisphenol A (BPA), o,p'-DDT (DDT), p-tert-octylphenol (OCT) and daidzein (DAI). A significant stimulation of the uterine wet weight could be observed after administration of all the substances. The activity of all analyzed compounds to stimulate uterine weight was low in comparison to EE. DDT has the highest activity to stimulate uterine weight whereas BPA and DAI turned out to be less potent. The analysis of gene expression revealed a very specific profile of molecular action in response to the different compounds which cannot be detected by judging the uterotropic response alone. A dose dependent analysis revealed that C3 mRNA is already modulated at doses where no uterotropic response was detectable. Although DAI and BPA were very weak stimulators of uterine growth, these substances were able to alter the expression of AR, ER and C3 very strongly. Based on these investigations the analyzed compounds can be subdivided into distinct classes: First, compounds which exhibit a similar gene expression fingerprint as EE (e.g. OCT); second, compounds exhibiting a significant uterotropic activity, but inducing a pattern of gene expression different from EE (e.g. DDT); and third, compounds like BPA and especially DAI which exhibit a very low uterotropic activity, but nevertheless modulate the expression of estrogen-sensitive genes. These findings strongly suggest that the fingerprint of uterine gene expression is a very sensitive tool to investigate estrogenicity of natural and synthetic compounds and offers the possibility to get information in regard to the molecular mechanisms involved in the action of the respective compounds.

Cloning of PRV-1, a novel member of the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera.

Polycythemia vera (PV) is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. Although it has been shown that progenitor cells of patients with PV are hypersensitive to several growth factors, the molecular pathogenesis of this disease remains unknown. To investigate the molecular defects underlying PV, we used subtractive hybridization to isolate complementary DNAs (cDNAs) differentially expressed in patients with PV versus normal controls. We isolated a novel gene, subsequently named PRV-1, which is highly expressed in granulocytes from patients with PV (n = 19), but not detectable in normal control granulocytes (n = 21). Moreover, PRV-1 is not expressed in mononuclear cells from patients with chronic myelogenous leukemia (n = 4) or acute myelogenous leukemia (n = 5) or in granulocytes from patients with essential thrombocythemia (n = 4) or secondary erythrocytosis (n = 4). Northern blot analysis showed that PRV-1 is highly expressed in normal human bone marrow and to a much lesser degree in fetal liver. It is not expressed in a variety of other tissues tested. Although PRV-1 is not expressed in resting granulocytes from normal controls, stimulation of these cells with granulocyte colony-stimulating factor induces PRV-1 expression. The PRV-1 cDNA encodes an open reading frame of 437 amino acids, which contains a signal peptide at the N-terminus and a hydrophobic segment at the C-terminus. In addition, PRV-1 contains 2 cysteine-rich domains homologous to those found in the uPAR/Ly6/CD59/snake toxin-receptor superfamily. We therefore propose that PRV-1 represents a novel hematopoietic receptor. (Blood. 2000;95:2569-2576)

Extracellular matrix regulates steady-state mRNA levels of the proliferation associated protein Ki-67 in endometrial cancer cells.

We investigated whether components of the extracellular matrix have the potential to regulate the proliferative activity of endometrial adenocarcinoma cells. Culturing of cells on the reconstituted basement membrane matrigel down-regulated the steady-state mRNA levels of the proliferation associated protein, Ki-67, in the endometrial adenocarcinoma cell lines HEC 1B(L) and Ishikawa after 48-96 h of culture on the matrix substrate. Proliferation of Ishikawa was stimulated again if cells were cultured on matrigel and challenged by proteins representing functional domains of tenascin-C, a mesenchymal glycoprotein. The fibronectin-type-III-like repeats 6-8 of tenascin-C were found to be the most potent. In summary, evidence is provided that components of both epithelial and stromal extracellular matrices can function as regulators of cell growth.

Characterization of estrogenicity of phytoestrogens in an endometrial-derived experimental model.

Severe developmental and reproductive disorders in wild animals have been linked to high exposure to persistent environmental chemicals with hormonal activity. These adverse effects of environmental estrogens have raised considerable concern and have received increasing attention. Although numerous chemicals with the capacity to interfere with the estrogen receptor (ER) have been identified, information on their molecular mechanism of action and their relative potency is rather limited. For the endometrium, the lack of information is due to the lack of a suitable experimental model. We investigated the functions of phytoestrogens in an endometrial-derived model, RUCA-I rat endometrial adenocarcinoma cells. The cells were cultured on a reconstituted basement membrane to preserve their functional differentiation and estrogen responsiveness. We assessed the relative binding affinity to the estrogen receptor of the selected phytoestrogens coumestrol, genistein, daidzein, and the putative phytoestrogen mangostin compared to estradiol by a competitive Scatchard analysis. The following affinity ranking was measured: 17beta-estradiol >>> coumestrol > genistein > daidzein >>> mangostin. In addition, we investigated the capacity of these compounds to promote the increased production of complement C3, a well-known estradiol-regulated protein of the rat endometrium. All substances tested increased the production of complement C3, although different concentrations were necessary to achieve equivalent levels of induction compared to estradiol. Mechanistically we were able to demonstrate that the increase of complement C3 production was mediated by primarily increasing its steady-state mRNA level. These findings indicate that RUCA-I cells represent a sensitive model system to elucidate relative potencies and functions of environmental estrogens in an endometrium-derived model.

A novel basement membrane-induced gene identified in the human endometrial adenocarcinoma cell line HEC1B.

Cultured HEC1B human endometrial adenocarcinoma cells respond to reconstituted basement membrane (Matrigel) by morphological and functional differentiation in vitro. Our goal is to identify genes involved in this differentiation process. By means of rt-PCR, we were able to isolate the novel 2.4 kb Matrigel-induced transcript icb-1 containing an open reading frame predicting a 31.7 kDa protein. The time-dependent induction of icb-1 gene expression by basement membrane was confirmed by Northern blot experiments. In a data bank search, several EST homologues corresponding to the 3' untranslated region could be found. In summary, icb-1 as a new tool enables us to study molecular mechanisms of cell-matrix interactions contributing to carcinogenesis.

Estrogen-dependent and cell-specific regulation of gene expression in RUCA-I endometrial adenocarcinoma cells.

Estrogens are believed to play a crucial role in growth regulation and differentiation of the normal endometrial tissue as well as in the carcinogenesis of the endometrium. Therefore, the influence of estrogens and antiestrogens on gene expression in the estrogen receptor-positive rat endometrial adenocarcinoma cell line RUCA-I was investigated. Differentially expressed genes were detected by differential display PCR of RNA of untreated, estradiol-treated and antiestrogen-treated RUCA-I cells. By means of the PCR technique, 14 differentially expressed fragments could be detected. Three of these 14 differentially expressed fragments were confirmed by Northern blotting. The steady state mRNA levels of the three gene fragments named AH41, AH42 and AH44 were downregulated by the antiestrogen ICI 164384. Further characterization revealed that the fragment AH41 is not expressed in stromal cells but in the human and rodent epithelial cell lines, BG-1 and RUCA-II. A comparison of the cDNA sequence of fragment AH41 with the EMBL database showed no high homology to known genes. Therefore, fragment AH41 has to be regarded as a fragment of a novel, estradiol-sensitive gene.

Basement membrane regulates gene expression in HEC1B(L) endometrial adenocarcinoma cells.

The morphology and differentiation of human endometrial adenocarcinoma cells HEC1B(L) are influenced by cell-matrix interactions. Culturing of HEC1B(L) cells on an extracellular matrix (ECM) induced the formation of a three-dimensionally arranged, highly ordered branching network of HEC1B(L) cells. In an effort to identify biologically important genes that are involved in this in vitro differentiation process of endometrial tumor cells we applied the method of "differential display." We isolated two DNA fragments, which were differentially regulated by ECM. One of these fragments, MESR4/13, was downregulated by ECM and contains a TG rich sequence. The second fragment, MES13/15, demonstrates high sequence similarity to MESR4/13 except for the TG-rich region and is induced by ECM. Probably these genes are potent markers to study the molecular basis of the process of in vitro differentiation of endometrial adenocarcinoma cells.

Variants of integrin beta 4 subunit in human endometrial adenocarcinoma cells: mediators of ECM-induced differentiation?

The influence of extracellular matrix (ECM) on expression and function of integrins in carcinogenesis and differentiation is not well understood, but the importance of altered adhesion features for tumor development and progression is obvious. Integrins as versatile molecules are mainly responsible for mediating cell-matrix interactions and transmembrane signal transduction. They are capable of transducing outside-in signals from ECM components or conversely to organize the matrix by inside-out signaling. In the study presented here, we report that the reconstituted basement membrane, Matrigel, which induces morphological and functional differentiation of the endometrial adenocarcinoma cell line HEC 1B(L), also regulates the expression of various forms of the integrin beta 4 subunit. Furthermore, we were able to identify full-length isoforms with and without an altered cytoplasmic domain as well as truncated forms. Our findings suggest a regulatory role of integrin beta 4 isoforms and fragments in the process of in vitro differentiation of HEC 1B(L).

Laminin mediates basement membrane induced differentiation of HEC 1B endometrial adenocarcinoma cells.

In vitro studies on endometrial carcinogenesis have been hampered by limited differentiation of the cells in culture. Using the endometrial carcinoma cell lines HEC 1B and its subclone HEC 1B(L), we established and characterized cell culture conditions that preserve a more differentiated state of the tumor cells. Randomly seeded HEC 1B(L) cells, if grown in a serum-free defined medium on top of a reconstituted basement membrane (Matrigel), within a few hours assembled themselves to web-like structures. In a thick layer of Matrigel, they showed an even more pronounced morphological differentiation. Functionally, two additional secretory proteins, about 31 and 77 kDa in size, became apparent as a response to matrigel. To further investigate the regulatory role of the extracellular matrix in the process of in vitro differentiation of endometrial adenocarcinoma cells, we addressed two specific problems. First, we investigated if the capacity of in vitro differentiation is a specific feature of HEC 1B(L) cells or if it is common to all endometrial adenocarcinoma cells. Second, we tried to identify the Matrigel component(s) responsible for in vitro differentiation. The assembly of HEC 1B and HEC 1B(L) cells into spatially organized web-like structures and the expression of the 77 kDa protein were thereby used as an assay. All endometrial adenocarcinoma cell lines tested to a variable degree formed web-like structures on Matrigel. Although the pattern of de novo synthesized secretory proteins changed as a response to Matrigel, only HEC 1A, HEC 1B, HEC 1B(L), and Ishikawa cells responded to culture on Matrigel by an increased expression of the 77 kDa protein. Functionally, polyclonal anti-laminin antibodies, but not anti-collagen type IV antibodies, disrupted formation of web-like structures by HEC 1B cells. The laminin-specific peptides YIGSR and SIKVAV but none of the RGD-peptides RGDS, GRGDSP, or GRADSP affected the three-dimensional assembly of these cells in vitro. Both anti-laminin antibodies and laminin-specific peptides suppressed Matrigel-induced formation of the 77-kDa secretory protein by HEC 1B cells. These findings suggest the involvement of laminin in the in vitro differentiation of the HEC 1B endometrial adenocarcinoma cell line. In a mechanistic view, laminin appears to play a crucial role in the regulation of this in vitro differentiation process.