MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERα) in regulation of the MUC1 gene expression

BACKGROUND: The MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter. RESULTS: Using TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERα, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed. CONCLUSION: Analysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERα in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy

Evidence for bystander signalling between human trophoblast cells and human embryonic stem cells

Maternal exposure during pregnancy to toxins can occasionally lead to miscarriage and malformation. It is currently thought that toxins pass through the placental barrier, albeit bilayered in the first trimester, and damage the fetus directly, albeit at low concentration. Here we examined the responses of human embryonic stem (hES) cells in tissue culture to two metals at low concentration. We compared direct exposures with indirect exposures across a bi-layered model of the placenta cell barrier. Direct exposure caused increased DNA damage without apoptosis or a loss of cell number but with some evidence of altered differentiation. Indirect exposure caused increased DNA damage and apoptosis but without loss of pluripotency. This was not caused by metal ions passing through the barrier. Instead the hES cells responded to signalling molecules (including TNF-α) secreted by the barrier cells. This mechanism was dependent on connexin 43 mediated intercellular ‘bystander signalling’ both within and between the trophoblast barrier and the hES colonies. These results highlight key differences between direct and indirect exposure of hES cells across a trophoblast barrier to metal toxins. It offers a theoretical possibility that an indirectly mediated toxicity of hES cells might have biological relevance to fetal development

Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells

<p>Abstract</p> <p>Background</p> <p>Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines.</p> <p>Methods</p> <p>Cultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis.</p> <p>Results</p> <p>We show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis.</p> <p>Conclusions</p> <p>The recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first evidence that altered primary cilium expression and function may be part of some malignant phenotypes. Further, we provide the first evidence that ciliogenesis is not an all or none process; rather defects can arrest this process at various points, particularly at the stage subsequent to basal body association with the plasma membrane.</p

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands

The dynamic cilium in human diseases

Cilia are specialized organelles protruding from the cell surface of almost all mammalian cells. They consist of a basal body, composed of two centrioles, and a protruding body, named the axoneme. Although the basic structure of all cilia is the same, numerous differences emerge in different cell types, suggesting diverse functions. In recent years many studies have elucidated the function of 9+0 primary cilia. The primary cilium acts as an antenna for the cell, and several important pathways such as Hedgehog, Wnt and planar cell polarity (PCP) are transduced through it. Many studies on animal models have revealed that during embryogenesis the primary cilium has an essential role in defining the correct patterning of the body. Cilia are composed of hundreds of proteins and the impairment or dysfunction of one protein alone can cause complete loss of cilia or the formation of abnormal cilia. Mutations in ciliary proteins cause ciliopathies which can affect many organs at different levels of severity and are characterized by a wide spectrum of phenotypes. Ciliary proteins can be mutated in more than one ciliopathy, suggesting an interaction between proteins. To date, little is known about the role of primary cilia in adult life and it is tempting to speculate about their role in the maintenance of adult organs. The state of the art in primary cilia studies reveals a very intricate role. Analysis of cilia-related pathways and of the different clinical phenotypes of ciliopathies helps to shed light on the function of these sophisticated organelles. The aim of this review is to evaluate the recent advances in cilia function and the molecular mechanisms at the basis of their activity

<i>Islet1</i> and Its Co-Factor <i>Ldb1</i> Are Expressed in Quiescent Cells of Mouse Intestinal Epithelium

<div><p><i>Islet1</i> belongs to Lim homeobox (<i>Lhx</i>) gene family which encodes transcription factors that have been conserved in evolution. They form complexes with other transcriptional regulators, among them obligatory co-factors encoded by <i>Ldb</i> genes. <i>Isl1 (Islet1)</i>, <i>Lhx</i> and <i>Ldb1</i> genes play a crucial role in organ patterning, cell fate determination and cell differentiation in both embryonic and adult tissues. In this study we analyzed expression pattern of <i>Isl1</i> and its co-factor <i>Ldb1</i> in small intestine. We also studied the biological role of <i>Ldb1</i> in gut endoderm. Quantitative PCR analysis revealed a relatively high level of expression of <i>Lhx1, Isl1, Isl2, Lmx1a, Ldb1</i> and <i>Ldb2</i> mRNAs in the gut tissue as compared to the level of less abundant detectable <i>Lmx1b</i> mRNA. Immunohistochemical studies demonstrated a unique pattern of Ldb1 and Islet1 proteins in the crypt compartment. Ldb1 is produced at a low level in majority of crypt cells; but, its abundant expression was demonstrated for some single cells. Islet1 is also expressed in single cells of the crypt. Double staining experiments with Ldb1 and Isl1 antibodies showed that both genes are co-expressed in certain cells of the crypt. Further analysis revealed the Ldb1-expressing cells in the gut are both of endodermal and mesodermal origin. Proliferation studies using antibodies to phospho-histone H3 and Ki-67 antigens, as well as long-term BrdU labeling, showed that cells prominently expressing Ldb1/Islet1 are quiescent but do not belong to any known terminally differentiated cell lineages. They may represent a group of stem-like cells in the crypt. Further experiments by cell lineage tracing should be performed to better characterize this cell population. Functional studies of mice with <i>Ldb1</i> gene ablated in gut endoderm revealed no specific role of <i>Ldb1</i> in that tissue.</p></div

Quantitative PCR analysis of mRNA expression of the LIM homeobox genes and their cofactors.

<p>The <i>Lhx1</i>, <i>Isl1</i>, <i>Isl2</i>, <i>Lmx1a</i>, <i>Lmx1b</i>, <i>Ldb1</i> and <i>Ldb2</i> mRNAs have been detected in mouse small intestine. <i>Lhx1</i>, <i>Islet1</i>, <i>Ldb1</i> and <i>Ldb2</i> mRNAs were expressed at high level. <i>Lhx2</i>, <i>Lhx3</i>, <i>Lhx4</i>, <i>Lhx5</i>, <i>Lhx6</i> and <i>Lhx8</i> mRNAs were not detected. All values are in log2 scale and were normalized to the level of <i>Lmx1b</i> mRNA which is considered 1. At least three independent experiments were performed for each gene. The error bars show standard deviations.</p

Localization of Ldb1- and Isl1-expressing cells in small intestine.

<p>Low level of Ldb1 protein is detected in most cells of the crypt (b and c). In a few single cells Ldb1 is expressed abundantly (arrows in b and c). Isl1 expression is observed in single cells of the proliferative zone of the crypt (arrowheads in e and f). No cells producing Ldb1 and Isl1 are found in the villous epithelium (data not shown). <b>a</b> and <b>d</b>. Negative control with pre-immune serum (x400). <b>b</b>. Expression of Ldb1 protein in the crypt (x400). <b>c</b>. High magnification of the selected area from <b>b</b> (x1000). Arrows point on cells which are highly expressing Ldb1. <b>e</b>. Expression of Isl1 protein in the crypt (x400). <b>f</b>. High magnification of the selected area from <b>e</b> (x1000). Arrowheads show cells which are prominently expressing Isl1. <b>g-i</b>. Co-localization of Ldb1 and Isl1 (x1000). Ldb1 (green) and Isl1 (red) are co-expressed (yellow) in single cells of the crypt. <b>g</b>. Ldb1 expression, <b>h</b>. Isl1 expression. <b>i</b>. Merge of <b>g</b> and <b>h</b>. Arrow pointed on cell which is co-expressing Ldb1 and Isl1. <b>j</b>. Location of Ldb1^high cells in the crypt. Ldb1^high cells occupied predominately position +4–+7.</p