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Sample records for breast epithelial cell

  1. Nerve Invasion by Epithelial Cells in Benign Breast Diseases

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    Yu-Jan Chan

    2009-03-01

    Full Text Available Nerve invasion by glandular epithelial cells in a lesion is usually regarded as invasive carcinoma. However, some benign conditions in the pancreas, prostate, breast and other organs may show involvement of nerve bundles by benign epithelial cells. We report an 18-year-old female with nerve invasion in benign breast disease. The lesion in her right breast revealed fibrocystic changes with ductal hyperplasia and stromal sclerosis. Perineural and intraneural involvement by bland-looking small ducts lined by 2 layers of cells including an outer layer of myoepithelial cells were found, suggestive of benign nerve invasion. There was no evidence of malignant cells in any of the sections. The patient remains well after 31 months of follow-up. About 44 cases of nerve invasion in benign breast diseases have been reported in the literature. It is necessary to carefully evaluate nerve involvement in breast lesions to avoid over-diagnosis and inappropriate operation.

  2. On the Sulfation and Methylation of Catecholestrogens in Human Mammary Epithelial Cells and Breast Cancer Cells

    National Research Council Canada - National Science Library

    Hui, Ying; Yasuda, Shin; Liu, Ming-Yih; Wu, Yi-yong; Liu, Ming-Cheh

    2008-01-01

    .... The present study was designed to examine the role of sulfation in the metabolism of CEs. MCF-7 breast cancer cells and MCF 10A human mammary epithelial cells were metabolically labeled with [35S...

  3. Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues.

    Science.gov (United States)

    Sasaki, M; Peterson, J A; Ceriani, R L

    1981-02-01

    A sensitive radioimmunoassay technique was developed to quantitate the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by absorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10(6) cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10(6) cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10(6) cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface.

  4. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

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    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  5. The role of epithelial cell adhesion molecule N-glycosylation on apoptosis in breast cancer cells.

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    Zhang, Dandan; Liu, Xue; Gao, Jiujiao; Sun, Yan; Liu, Tingjiao; Yan, Qiu; Yang, Xuesong

    2017-03-01

    Glycosylation of cell surface proteins plays an important role in the regulation of apoptosis. It has been demonstrated that knockdown of epithelial cell adhesion molecule promoted apoptosis, inhibited cell proliferation, and caused cell-cycle arrest. In this study, we investigated whether and how N-glycosylation of epithelial cell adhesion molecule influenced the apoptosis in breast cancer cells. We applied the N-glycosylation mutation epithelial cell adhesion molecule plasmid to express deglycosylation of epithelial cell adhesion molecule and then to study its function. Our results showed that deglycosylation of epithelial cell adhesion molecule promoted apoptosis and inhibited cell proliferation. Deglycosylation of epithelial cell adhesion molecule enhanced the cytotoxic effect of 5-fluorouracil, promoting apoptosis by downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of the pro-apoptotic proteins Bax and Caspase 3 via the extracellular-signal-regulated kinase 1/2 and c-Jun N-terminal kinase mitogen-activated protein kinase signaling pathways in MCF-7 and MDA-MB-231 cells. These findings are important for a better understanding of epithelial cell adhesion molecule apoptosis regulation and suggest epithelial cell adhesion molecule as a potential target for the treatment of breast cancer.

  6. MUC-1-/ESA+ progenitor cells in normal, benign and malignant human breast epithelial cells.

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    Lü, Xinquan; Li, Huixiang; Xu, Kejia; Nesland, Jahn M; Suo, Zhenhe

    2009-11-01

    The existence of mammary epithelial stem/progenitor cells has been demonstrated in MUC-1-/ESA+ subpopulations of breast epithelial cells. However, knowledge about the expression and localization in benign and malignant breast lesions is unknown. Using a double-staining immunohistochemistry method, we investigated MUC-1-/ESA+ cells in 10 normal breast tissues, 49 cases with fibrocystic disease, 40 fibroadenomas, 36 invasive ductal carcinomas and the breast cancer cell lines MCF-7 and MDA-MB-468. In normal breast tissues MUC-1-/ESA+ cells were mainly found in the suprabasal layer, but under the apical surface of the duct/alveolus. In the hyperplastic areas of fibrocystic disease, the number of this subpopulation of cells was higher than that in hypoplastic areas and in fibroadenomas. In invasive ductal carcinoma, the MMUC-1-/ESA+ cells were heterogeneously present in different carcinoma nests. In the MCF-7 cell line most cells were MUC-1-/ESA+, and in the MDA-MB-468 cell line MUC-1-/ESA+ cells and MUC-1-/ESA+ cells were almost equal. Our results show that the MUC-1-/ESA+ subpopulation increases in fibrocystic disease within the hyperplastic areas, and varies in benign and malignant breast tumours, indicating that breast carcinogenesis may develop from malignant changes of normal MUC-1-/ESA+ cells.

  7. Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells

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    Vaapil, Marica; Helczynska, Karolina; Villadsen, René

    2012-01-01

    Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less...... is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis....

  8. The effect of hydroxybenzoate calcium compounds in inducing cell death in epithelial breast cancer cells

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    Nada M Merghani

    2015-12-01

    Full Text Available Hydroxybenzoate (HB compounds have shown their significance in inducing apoptosis in primary chronic lymphocytic leukemia (CLL and cancer cell lines, including HT-1080. The current study focuses on assessing the effects of 2-, 3- and 4-hydroxybenzoate calcium (HBCa compounds on MCF-10A, MDA-MB231 and MCF-7 epithelial breast cell lines. The HBCa-treated cells were examined using annexin V, to measure apoptosis in the three epithelial breast cell lines, after 48 h of treatment. The results indicated that 0.5 and 2.5 mmol/L of HBCa induced cell death in a dose-dependent manner. The induction of cell death in normal MCF-10A cells was found to be significantly less (p = 0.0003–0.0068, in comparison to the malignant cell lines (MDA-MB231 and MCF-7. HBCa compounds were also found to cause cell cycle arrest in the epithelial breast cells at G1/G0. Furthermore, HBCa compounds induced the upregulation of apoptotic proteins (p53, p21, Bax and caspase-3, as well as the downregulation of the anti-apoptotic protein Bcl-2, which may suggest that apoptosis is induced via the intrinsic pathway.

  9. Effects of Radiation Therapy on Breast Epithelial Cells in BRCA1/2 Mutation Carriers.

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    Chiang, Huai-Chin; Elledge, Richard; Larson, Paula; Jatoi, Ismail; Li, Rong; Hu, Yanfen

    2015-01-01

    Women carrying BRCA1 and BRCA2 mutations have significantly elevated risk of developing breast and ovarian cancers. BRCA1-associated breast cancer likely originates from progenitors of the luminal epithelial lineage. Recent studies indicate that radiation therapy (RT) for BRCA1 cancer patients is associated with lower incidence of developing subsequent ipsilateral breast cancer. In the current study, we analyzed tumor-free breast tissue procured via prophylactic bilateral mastectomy from three BRCA1 and one BRCA2 mutation carriers, who had been previously treated with RT for unilateral breast cancers. Freshly isolated breast cells from the irradiated and nonirradiated breast tissue of the same individuals were subjected to flow cytometry, using established cell-surface markers. Two out of the three BRCA1 carriers and one BRCA2 carrier exhibited significantly diminished luminal cell population in the irradiated breast versus the nonirradiated side. There was also RT-associated reduction in the colony-forming ability of the breast epithelial cells. Our finding suggests that prior RT could result in the depletion of the luminal epithelial compartment and thus reduced incidence of BRCA1/2-associated breast cancer.

  10. Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia

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    Petersen, Ole William; Gudjonsson, Thorarinn; Villadsen, René

    2003-01-01

    The majority of human breast carcinomas exhibit luminal characteristics and as such, are most probably derived from progenitor cells within the luminal epithelial compartment. This has been subdivided recently into at least three luminal subtypes based on gene expression patterns. The value of kn......% of breast cancers arise in TDLUs and more than 90% are also cytokeratin 19-positive, we suggest that this cell population contains a breast-cancer progenitor.......The majority of human breast carcinomas exhibit luminal characteristics and as such, are most probably derived from progenitor cells within the luminal epithelial compartment. This has been subdivided recently into at least three luminal subtypes based on gene expression patterns. The value...... of knowing the cellular origin of individual tumours is clear and should aid in designing effective therapies. To do this, however, we need strategies aimed at defining the nature of stem and progenitor cell populations in the normal breast. In this review, we will discuss our technical approach...

  11. Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

    DEFF Research Database (Denmark)

    Roslind, A.; Balslev, E.; Kruse, H.

    2008-01-01

    . YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial......YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy...

  12. The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

    Energy Technology Data Exchange (ETDEWEB)

    Petersen, Ole William; Nielsen, Helga Lind; Gudjonsson, Thorarinn; Villadsen, Ren& #233; ; Ronnov-Jessen, Lone; Bissell, Mina J.

    2001-05-12

    The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neoplastic breast epithelial cells may be substantially more plastic in their differentiation repertoire than previously anticipated. Thus, along with an increasing availability of markers for the myoepithelial lineage, at least a partial differentiation towards this lineage is being revealed frequently. It has also become clear that conversions towards the mesenchymal lineage actually occur, referred to as epithelial to mesenchymal transitions. Indeed, some of the so-called myofibroblasts surrounding the tumor may indeed have an epithelial origin rather than a mesenchymal origin. Because myoepithelial cells, epithelial to mesenchymal transition-derived cells, genuine stromal cells and myofibroblasts share common markers, we now need to define a more ambitious set of markers to distinguish these cell types in the microenvironment of the tumors. This is necessary because the different microenvironments may confer different clinical outcomes. The aim of this commentary is to describe some of the inherent complexities in defining cellular phenotypes in the microenvironment of breast cancer and to expand wherever possible on the implications for tumor suppression and progression.

  13. Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells.

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    Marica Vaapil

    Full Text Available INTRODUCTION: Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. METHODS: Normal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot. RESULTS: In large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar

  14. CDDO-Me protects normal lung and breast epithelial cells but not cancer cells from radiation.

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    El-Ashmawy, Mariam; Delgado, Oliver; Cardentey, Agnelio; Wright, Woodring E; Shay, Jerry W

    2014-01-01

    Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs). In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF) = 1.3), and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs) with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients.

  15. Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer.

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    Zhang, Le; Xu, Liang; Zhang, Fengchun; Vlashi, Erina

    2017-04-18

    Experimental evidence suggest that breast tumors originate from breast cancer stem cells (BCSCs), and that mitochondrial biogenesis is essential for the anchorage-independent clonal expansion and survival of CSCs, thus rendering mitochondria a significant target for novel treatment approaches. One of the recognized side effects of the FDA-approved drug, doxycycline is the inhibition of mitochondrial biogenesis. Here we investigate the mechanism by which doxycycline exerts its inhibitory effects on the properties of breast cancer cells and BCSCs, such as mammosphere forming efficiency, invasion, migration, apoptosis, the expression of stem cell markers and epithelial-to-mesenchymal transition (EMT) related markers of breast cancer cells. In addition, we explored whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells. We find that doxycyline can inhibit the viability and proliferation of breast cancer cells and BCSCs, decrease mammosphere forming efficiency, migration and invasion, and EMT of breast cancer cells. Expression of stem cell factors Oct4, Sox2, Nanog and CD44 were also significantly downregulated after doxycycline treatment. Moreover, doxycycline could down-regulate the expression of the autophagy marker LC-3BI and LC-3BII, suggesting that inhibiting autophagy may be responsible in part for the observed effects on proliferation, EMT and stem cell markers. The potent inhibition of EMT and cancer stem-like characteristics in breast cancer cells by doxycycline treatment suggests that this drug can be repurposed as an anti-cancer drug in the treatment of breast cancer patients in the clinic.

  16. Neoplastic transformation of breast epithelial cells by genotoxic stress

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    Raman Venu

    2010-06-01

    Full Text Available Abstract Background Exposure to genotoxic stresses such as radiation and tobacco smoke can cause increased cancer incidence rate as reflected in an in depth meta-analysis of data for women and breast cancer incidence. Published reports have indicated that exposures to low dose radiation and tobacco smoke are factors that contribute to the development of breast cancer. However, there is a scarcity of information on the combinatorial effects of low dose radiation and tobacco smoke on formation and progression of breast cancer. The combination of these two genotoxic insults can induce significant damage to the genetic material of the cells resulting in neoplastic transformation. Methods To study the effects of low dose ionizing radiation and tobacco smoke on breast cells, MCF 10A cells were treated either with radiation (Rad - 0.1 Gray or cigarette smoke condensate (Csc - 10 microgram/ml of medium or a combination of Rad + Csc. Following treatments, cells were analyzed for cell cycle distribution patterns and the ability to extrude the Hoechst 33342 dye. In addition, in vitro invasion and migration as well as mammosphere formation assays were performed. Finally, differential gene expression profiles were generated from the individual and combination treatment. Results Exposure of MCF 10A cells to the combination of radiation plus cigarette smoke condensate generated a neoplastic phenotype. The transformed phenotype promoted increased mammosphere numbers, altered cell cycle phases with a doubling of the population in S phase, and increased invasion and motility. Also, exclusion of Hoechst 33342 dye, a surrogate marker for increased ABC transporters, was observed, which indicates a possible increase in drug resistance. In addition, changes in gene expression include the up regulation of genes encoding proteins involved in metabolic pathways and inflammation. Conclusions The results indicate that when normal breast cells are exposed to low dose

  17. Neoplastic transformation of breast epithelial cells by genotoxic stress.

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    Botlagunta, Mahendran; Winnard, Paul T; Raman, Venu

    2010-06-30

    Exposure to genotoxic stresses such as radiation and tobacco smoke can cause increased cancer incidence rate as reflected in an in depth meta-analysis of data for women and breast cancer incidence. Published reports have indicated that exposures to low dose radiation and tobacco smoke are factors that contribute to the development of breast cancer. However, there is a scarcity of information on the combinatorial effects of low dose radiation and tobacco smoke on formation and progression of breast cancer. The combination of these two genotoxic insults can induce significant damage to the genetic material of the cells resulting in neoplastic transformation. To study the effects of low dose ionizing radiation and tobacco smoke on breast cells, MCF 10A cells were treated either with radiation (Rad - 0.1 Gray) or cigarette smoke condensate (Csc - 10 microgram/ml of medium) or a combination of Rad + Csc. Following treatments, cells were analyzed for cell cycle distribution patterns and the ability to extrude the Hoechst 33342 dye. In addition, in vitro invasion and migration as well as mammosphere formation assays were performed. Finally, differential gene expression profiles were generated from the individual and combination treatment. Exposure of MCF 10A cells to the combination of radiation plus cigarette smoke condensate generated a neoplastic phenotype. The transformed phenotype promoted increased mammosphere numbers, altered cell cycle phases with a doubling of the population in S phase, and increased invasion and motility. Also, exclusion of Hoechst 33342 dye, a surrogate marker for increased ABC transporters, was observed, which indicates a possible increase in drug resistance. In addition, changes in gene expression include the up regulation of genes encoding proteins involved in metabolic pathways and inflammation. The results indicate that when normal breast cells are exposed to low dose radiation in combination with cigarette smoke condensate a phenotype is

  18. Myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and stalls invasion.

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    Ruth Li

    Full Text Available Myoferlin (MYOF is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET. These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNA(MYOF transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF.

  19. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

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    Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

    2012-12-01

    The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

  20. Prepubertal unilateral gynecomastia and the presence of 47,XXY mosaicism in breast epithelial cells

    DEFF Research Database (Denmark)

    Andersen, Peter Stemann; Petersen, Bodil Laub; Juul, Anders

    2013-01-01

    , and sclerotherapy with Picibanil (OK-432) was attempted without any detectable effect on size. The mass was later excised. The pathological examination revealed mammary gland tissue suggestive of idiopathic gynecomastia. FISH revealed 47, XXY mosaicism in the abnormal breast epithelial cells, but not in peripheral...

  1. Breast Cancer Stem Cells Transition between Epithelial and Mesenchymal States Reflective of their Normal Counterparts

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    Suling Liu

    2014-01-01

    Full Text Available Previous studies have suggested that breast cancer stem cells (BCSCs mediate metastasis, are resistant to radiation and chemotherapy, and contribute to relapse. Although several BCSC markers have been described, it is unclear whether these markers identify the same or independent BCSCs. Here, we show that BCSCs exist in distinct mesenchymal-like (epithelial-mesenchymal transition [EMT] and epithelial-like (mesenchymal-epithelial transition [MET] states. Mesenchymal-like BCSCs characterized as CD24−CD44+ are primarily quiescent and localized at the tumor invasive front, whereas epithelial-like BCSCs express aldehyde dehydrogenase (ALDH, are proliferative, and are located more centrally. The gene-expression profiles of mesenchymal-like and epithelial-like BCSCs are remarkably similar across different molecular subtypes of breast cancer, and resemble those of distinct basal and luminal stem cells found in the normal breast. We propose that the plasticity of BCSCs that allows them to transition between EMT- and MET-like states endows these cells with the capacity for tissue invasion, dissemination, and growth at metastatic sites.

  2. Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

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    Özel Cem

    2012-04-01

    Full Text Available Abstract Background The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-β/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. Results Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. Conclusions Here we show that hybrid cells could evolve exhibiting a

  3. BRCA1 promoter methylation of normal breast epithelial cells as a possible precursor for BRCA1-methylated breast cancer

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    Otani, Yoko; Miyake, Tomohiro; Kagara, Naofumi; Shimoda, Masafumi; Naoi, Yasuto; Maruyama, Naomi; Shimomura, Atsuhi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

    2014-01-01

    The breast cancer susceptibility gene 1 (BRCA1) and glutathione S-transferase P1 (GSTP1) promoters are reportedly often methylated in breast cancer tissues. Their methylation status in surrounding normal breast tissues has not been examined thoroughly although this may well be important for a better understanding of breast carcinogenesis. In this study, BRCA1 and GSTP1 promoter methylation was examined by methylation-specific PCR assay. Patients with BRCA1-methylated (n = 15) or BRCA1-unmethylated (n = 15) tumors and those with GSTP1-methylated (n = 9) or GSTP1-unmethylated (n = 11) tumors were included in the present study. Methylation status of manually micro-dissected normal epithelial cells from the formalin-fixed paraffin-embedded sections of normal breast tissues adjacent to and distant from the tumors was examined at multiple sites (n = 1–5). Of the 15 patients with BRCA1-methylated tumors, 9 harbored BRCA1 promoter methylation in at least one site of the normal breast tissues. However, no BRCA1 promoter methylation was observed at any site of the normal tissues of the 15 patients with BRCA1-unmethylated tumors. No GSTP1 promoter methylation was observed in the normal tissues regardless of the methylation status of the tumors. The presence of BRCA1 promoter methylation in the normal tissues was confirmed in the epithelial cells enriched with the magnetic-activated cell sorting method. Our findings suggest that a small proportion of normal breast epithelial cells with BRCA1 promoter methylation can be precursor cells from which BRCA1-methylated breast tumors may originate. This does not apply to GSTP1 promoter methylation. PMID:25155055

  4. Expression of TRPC6 channels in human epithelial breast cancer cells

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    Ahidouch Ahmed

    2008-05-01

    Full Text Available Abstract Background TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7, a human breast cancer epithelial primary culture (hBCE and in normal and tumour breast tissues. Methods Molecular (Western blot and RT-PCR, and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration. Results A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPC7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours. Conclusion Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

  5. DHEA increases epithelial markers and decreases mesenchymal proteins in breast cancer cells and reduces xenograft growth.

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    Colín-Val, Zaira; González-Puertos, Viridiana Yazmín; Mendoza-Milla, Criselda; Gómez, Erika Olivia; Huesca-Gómez, Claudia; López-Marure, Rebeca

    2017-10-15

    Breast cancer is one of the most common neoplasias and the leading cause of cancer death in women worldwide. Its high mortality rate is linked to a great metastatic capacity associated with the epithelial-mesenchymal transition (EMT). During this process, a decrease in epithelial proteins expression and an increase of mesenchymal proteins are observed. On the other hand, it has been shown that dehydroepiandrosterone (DHEA), the most abundant steroid in human plasma, inhibits migration of breast cancer cells; however, the underlying mechanisms have not been elucidated. In this study, the in vitro effect of DHEA on the expression pattern of some EMT-related proteins, such as E-cadherin (epithelial), N-cadherin, vimentin and Snail (mesenchymal) was measured by Western blot and immunofluorescence in MDA-MB-231 breast cancer cells with invasive, metastatic and mesenchymal phenotype. Also, the in vivo effect of DHEA on xenograft tumor growth in nude mice (nu(-)/nu(-)) and on expression of the same epithelial and mesenchymal proteins in generated tumors was evaluated. We found that DHEA increased expression of E-cadherin and decreased N-cadherin, vimentin and Snail expression both in MD-MB-231 cells and in the formed tumors, possibly by DHEA-induced reversion of mesenchymal phenotype. These results were correlated with a tumor size reduction in mouse xenografts following DHEA administration either a week earlier or concurrent with breast cancer cells inoculation. In conclusion, DHEA could be useful in the treatment of breast cancer with mesenchymal phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

    Science.gov (United States)

    Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

    2014-01-01

    Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

  7. Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells.

    Science.gov (United States)

    Khanna, Sugandha; Darbre, Philippa D

    2013-05-01

    Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Schedin Pepper J

    2010-07-01

    Full Text Available Abstract Background Recent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially immune and stromal cells, in the progression of cancer and the development of metastases. However, the role of adjacent benign epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. In this report we addressed the question whether benign MECs alter the transformed phenotype of human breast cancer cells. Methods We used both in vitro and in vivo co-cultivation approaches, whereby we mixed GFP-tagged MCF-10A cells (G2B-10A, as a model of benign mammary epithelial cells (MECs, and RFP-tagged MDA-MB-231-TIAS cells (R2-T1AS, as a model of breast cancer cells. Results The in vitro studies showed that G2B-10A cells increase the colony formation of R2-T1AS cells in both soft agar and clonogenicity assays. Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA fixation of G2B-10A cells abrogated this enhancement effect. Moreover, two other models of benign MECs, MCF-12A and HuMECs, also enhanced R2-T1AS colony growth in soft agar and clonogenicity assays. These data reveal that factors secreted by benign MECs are responsible for the observed enhancement of the R2-T1AS transformed phenotype. To determine whether G2B-10A cells enhance the tumorigenic growth of co-injected R2-T1AS cells in vivo, we used the nude mouse xenograft assay. Co-injecting R2-T1AS cells with G2B-10A cells ± PFA-fixation, revealed that G2B-10A cells promoted a ~3-fold increase in tumor growth, irrespective of PFA pre-treatment. These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay. Finally, using array analysis, we found that both live and PFA

  9. Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

    OpenAIRE

    Nakles, Rebecca E.; Millman, Sarah L.; Cabrera, M. Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S.; Schroeder, Timm; Furth, Priscilla A.

    2013-01-01

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without...

  10. Neoplastic transformation of breast epithelial cells by genotoxic stress

    OpenAIRE

    Raman Venu; Winnard Paul T; Botlagunta Mahendran

    2010-01-01

    Abstract Background Exposure to genotoxic stresses such as radiation and tobacco smoke can cause increased cancer incidence rate as reflected in an in depth meta-analysis of data for women and breast cancer incidence. Published reports have indicated that exposures to low dose radiation and tobacco smoke are factors that contribute to the development of breast cancer. However, there is a scarcity of information on the combinatorial effects of low dose radiation and tobacco smoke on formation ...

  11. Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells.

    Science.gov (United States)

    Pistone Creydt, Virginia; Fletcher, Sabrina Johanna; Giudice, Jimena; Bruzzone, Ariana; Chasseing, Norma Alejandra; Gonzalez, Eduardo Gustavo; Sacca, Paula Alejandra; Calvo, Juan Carlos

    2013-02-01

    Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

  12. Role of epigenetic mechanisms in epithelial-to-mesenchymal transition of breast cancer cells.

    Science.gov (United States)

    Nickel, Annina; Stadler, Sonja C

    2015-01-01

    The epithelial-to-mesenchymal transition (EMT) is a crucial process during normal development that allows dynamic and reversible shifts between epithelial and mesenchymal cell states. Cancer cells take advantage of the complex, interrelated cellular networks that regulate EMT to promote their migratory and invasive capabilities. During the past few years, evidence has accumulated that indicates that genetic mutations and changes to epigenetic mechanisms are key drivers of EMT in cancer cells. Recent studies have begun to shed light on the epigenetic reprogramming in cancer cells that enables them to switch from a noninvasive form to an invasive, metastatic form. The authors review the current knowledge of alterations of epigenetic machinery, including DNA methylation, histone modifications, nucleosome remodeling and expression of microRNAs, associated with EMT and tumor progression of breast cancer cells. Last, existing and upcoming drug therapies targeting epigenetic regulators and their potential benefit for developing novel treatment strategies are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Estrogen receptor silencing induces epithelial to mesenchymal transition in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Sanaa Al Saleh

    Full Text Available We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in trans-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII with altered morphology, increased motility with rearrangement and switch from a keratin/actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated geometric fold changes ≥ 3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. These data support our hypothesis that induced loss of estrogen receptor in previously estrogen/antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of

  14. All trans-retinoic acid (ATRA) induces re-differentiation of early transformed breast epithelial cells.

    Science.gov (United States)

    Arisi, Maria F; Starker, Rebecca A; Addya, Sankar; Huang, Yong; Fernandez, Sandra V

    2014-06-01

    Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic and antioxidant properties. We investigated the effect of all trans-retinoic acid (ATRA) at different stages of the neoplastic transformation using an in vitro model of breast cancer progression. This model was previously developed by treating the MCF-10F human normal breast epithelial cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: MCF-10F, normal stage; trMCF, transformed MCF-10F; bsMCF, invasive stage; and caMCF, tumorigenic stage. In 3D cultures, MCF-10F cells form tubules resembling the structures in the normal mammary gland. After treatment with estradiol, these cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with ATRA. After treatment with 10 or 1 µM ATRA, the trMCF clone 11 cells showed tubules in collagen; 10 and 43% of the structures were tubules in cells treated with 10 and 1 µM ATRA, respectively. Gene expression studies showed that 207 genes upregulated in transformed trMCF clone 11 cells were downregulated after 1 µM ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were downregulated in trMCF clone 11 were upregulated after 1 µM ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 1 µM ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after ATRA treatment. These results suggest that ATRA could be used as a chemopreventive agent to

  15. The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

    DEFF Research Database (Denmark)

    Petersen, Ole William; Nielsen, Helga Lind; Gudjonsson, Thorarinn

    2001-01-01

    The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neopl...

  16. CCL20 promotes migration and invasiveness of human cancerous breast epithelial cells in primary culture.

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Marsigliante, Santo

    2017-11-01

    The relation between the tumor and its microenvironment is one of the most interesting and less understood issues. Recently, we showed a role of CCL20 chemokine in proning the healthy tissue neighboring the tumor to carcinogenesis. Besides, tumor-secreted CCL20 induced proliferation, migration, and EMT of healthy cells. In this context, we have studied here if CCL20 had effects on the migration of cancer cells and the intracellular pathways used in breast epithelial cells in primary culture. Using molecular (siRNA) and pharmacological (inhibitors) techniques, we found multiple signaling kinases to be activated and involved in CCL20-induced tumor breast cell migration. CCL20 provoked a 2.5-fold increase of cell migration and invasion; CCL20 also enhanced MMP- 2 and MMP-9 mRNAs/protein expression and activities. Cell migration and invasiveness due to CCL20 significantly decreased when MMP-2 and MMP-9 were inhibited in CCL20-stimulated cells. CCL20 controlled MMP-2 expression through the JAK2/STAT3 pathway, while the expression of MMP-9 occurred by PKC-α that activated, consequently, c-Src, Akt, and finally NF-kB. These results reveal a role for CCL20 also in tumor breast cell and point to CCL20 as a novel therapeutic target in cancer. © 2017 Wiley Periodicals, Inc.

  17. Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement.

    Science.gov (United States)

    Dinicola, Simona; Fabrizi, Gianmarco; Masiello, Maria Grazia; Proietti, Sara; Palombo, Alessandro; Minini, Mirko; Harrath, Abdel Halim; Alwasel, Saleh H; Ricci, Giulia; Catizone, Angela; Cucina, Alessandra; Bizzarri, Mariano

    2016-07-01

    Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, β-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    Science.gov (United States)

    2014-01-01

    Background Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied. Methods Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay. Results We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti

  19. Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Laura Smith

    Full Text Available MicroRNA (miR expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells.We used laser microdissection (LMD to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERβ1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs and cancer-associated fibroblasts (CAFs from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01 and being lowest in invasive breast tissue (p<0.01. This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078 and invasive breast cancer (p = 0.031. ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells.miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional

  20. Lipopolysaccharide (LPS) Promotes Apoptosis in Human Breast Epithelial × Breast Cancer Hybrids, but Not in Parental Cells.

    Science.gov (United States)

    Fried, Sabrina; Tosun, Songuel; Troost, Gabriele; Keil, Silvia; Zaenker, Kurt S; Dittmar, Thomas

    2016-01-01

    Toll-like receptors (TLRs) belong to the group of pathogen recognition receptors known to play a crucial role in the innate immune system. In cancer, TLR expression is still debated controversially due to contradictory results reporting that both induction of apoptosis as well as tumor progression could depend on TLR signaling, whereby recent data rather indicate a pro-tumorigenic effect. The biological phenomenon of cell fusion has been associated with cancer progression due to findings revealing that fusion-derived hybrid cells could exhibit properties like an increased metastatogenic capacity and an increased drug resistance. Thus, M13MDA435 hybrid cell lines, which derived from spontaneous fusion events between human M13SV1-EGFP-Neo breast epithelial cells and human MDA-MB-435-Hyg breast cancer cells, were investigated. Cultivation of cells in the presence of the TLR4 ligand LPS potently induced apoptosis in all hybrid clones, but not in parental cells, which was most likely attributed to differential kinetics of the TLR4 signal transduction cascade. Activation of this pathway concomitant with NF-κB nuclear translocation and TNF-α expression was solely observed in hybrid cells. However, induction of LPS mediated apoptosis was not TNF-α dependent since TNF-α neutralization was not correlated to a decreased amount of dead cells. In addition to TNF-α, LPS also caused IFN-β expression in hybrid clones 1 and 3. Interestingly, hybrid clones differ in the mode of LPS induced apoptosis. While neutralization of IFN-β was sufficient to impair the LPS induced apoptosis in M13MDA435-1 and -3 hybrids, the amount of apoptotic M13MDA435-2 and -4 hybrid cells remained unchanged in the presence of neutralizing IFN-β antibodies. In summary, the fusion of non-LPS susceptible parental human breast epithelial cells and human breast cancer cells gave rise to LPS susceptible hybrid cells, which is in view with the cell fusion hypothesis that hybrid cells could exhibit novel

  1. Rap1 integrates tissue polarity, lumen formation, and tumorigenicpotential in human breast epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.; Bissell,Mina J.

    2006-09-29

    Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

  2. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent.

    Science.gov (United States)

    Davis, F M; Azimi, I; Faville, R A; Peters, A A; Jalink, K; Putney, J W; Goodhill, G J; Thompson, E W; Roberts-Thomson, S J; Monteith, G R

    2014-05-01

    Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of

  3. Molecular monitoring of epithelial-to-mesenchymal transition in breast cancer cells by means of Raman spectroscopy.

    Science.gov (United States)

    Marro, M; Nieva, C; Sanz-Pamplona, R; Sierra, A

    2014-09-01

    In breast cancer the presence of cells undergoing the epithelial-to-mesenchymal transition is indicative of metastasis progression. Since metabolic features of breast tumour cells are critical in cancer progression and drug resistance, we hypothesized that the lipid content of malignant cells might be a useful indirect measure of cancer progression. In this study Multivariate Curve Resolution was applied to cellular Raman spectra to assess the metabolic composition of breast cancer cells undergoing the epithelial to mesenchymal transition. Multivariate Curve Resolution analysis led to the conclusion that this transition affects the lipid profile of cells, increasing tryptophan but maintaining a low fatty acid content in comparison with highly metastatic cells. Supporting those results, a Partial Least Square-Discriminant analysis was performed to test the ability of Raman spectroscopy to discriminate the initial steps of epithelial to mesenchymal transition in breast cancer cells. We achieved a high level of sensitivity and specificity, 94% and 100%, respectively. In conclusion, Raman microspectroscopy coupled with Multivariate Curve Resolution enables deconvolution and tracking of the molecular content of cancer cells during a biochemical process, being a powerful, rapid, reagent-free and non-invasive tool for identifying metabolic features of breast cancer cell aggressiveness at first stages of malignancy. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. FGF10: Type III Epithelial Mesenchymal Transition and Invasion in Breast Cancer Cell Lines

    Science.gov (United States)

    Abolhassani, Ali; Riazi, Gholam Hossein; Azizi, Ebrahim; Amanpour, Saeid; Muhammadnejad, Samad; Haddadi, Mahnaz; Zekri, Ali; Shirkoohi, Reza

    2014-01-01

    Purpose: Fibroblastic growth factor-10 (FGF-10) has an important role in type I epithelial mesenchymal transition (EMT) during the embryonic period of life (gastrulation). Since EMT has a critical role during cancer cells invasion and metastasis (type III) this study sought to investigate the possible role of FGF-10 in type III EMT by monitoring breast cancer cell lines' behavior by FGF-10 regulation. Methods: MCF-7 and MDA-MB-231 cell lines with different levels of FGF10 expression were treated with FGF-10 recombinant protein and FGF-10 siRNA, respectively. Results: The cell viability, migration, colony formation and wound healing have a direct relationship with FGF-10 expression, while FGF-10 expression decreased apoptosis. All mesenchymal factors (such as vimentin, N cadherin, snail, slug, TGF-β) increased due to FGF-10 expression with contrary expression of epithelial markers (such as E-cadherin). Moreover, GSK3β phosphorylation (inactivation) increased with FGF-10 expression. Conclusion: The important role of FGF-10 in type III EMT on cancer cells and initiation of metastasis via various kinds of signaling pathways has been suggested. PMID:25057305

  5. Three-Dimensional Culture of Human Breast Epithelial Cells: The How and the Why

    Science.gov (United States)

    Vidi, Pierre-Alexandre; Bissell, Mina J.; Lelièvre, Sophie A.

    2013-01-01

    Organs are made of the organized assembly of different cell types that contribute to the architecture necessary for functional differentiation. In those with exocrine function, such as the breast, cell–cell and cell–extracellular matrix (ECM) interactions establish mechanistic constraints and a complex biochemical signaling network essential for differentiation and homeostasis of the glandular epithelium. Such knowledge has been elegantly acquired for the mammary gland by placing epithelial cells under three-dimensional (3D) culture conditions. Three-dimensional cell culture aims at recapitulating normal and pathological tissue architectures, hence providing physiologically relevant models to study normal development and disease. The specific architecture of the breast epithelium consists of glandular structures (acini) connected to a branched ductal system. A single layer of basoapically polarized luminal cells delineates ductal or acinar lumena at the apical pole. Luminal cells make contact with myoepithelial cells and, in certain areas at the basal pole, also with basement membrane (BM) components. In this chapter, we describe how this exquisite organization as well as stages of disorganization pertaining to cancer progression can be reproduced in 3D cultures. Advantages and limitations of different culture settings are discussed. Technical designs for induction of phenotypic modulations, biochemical analyses, and state-of-the-art imaging are presented. We also explain how signaling is regulated differently in 3D cultures compared to traditional two-dimensional (2D) cultures. We believe that using 3D cultures is an indispensable method to unravel the intricacies of human mammary functions and would best serve the fight against breast cancer. PMID:23097109

  6. Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li Qianqian

    2010-10-01

    Full Text Available Abstract Background Spontaneous immortalisation of cultured mammary epithelial cells (MECs is an extremely rare event, and the molecular mechanism behind spontaneous immortalisation of MECs is unclear. Here, we report the establishment of a spontaneously immortalised bovine mammary epithelial cell line (BME65Cs and the changes in gene expression associated with BME65Cs cells. Results BME65Cs cells maintain the general characteristics of normal mammary epithelial cells in morphology, karyotype and immunohistochemistry, and are accompanied by the activation of endogenous bTERT (bovine Telomerase Reverse Transcriptase and stabilisation of the telomere. Currently, BME65Cs cells have been passed for more than 220 generations, and these cells exhibit non-malignant transformation. The expression of multiple genes was investigated in BME65Cs cells, senescent BMECs (bovine MECs cells, early passage BMECs cells and MCF-7 cells (a human breast cancer cell line. In comparison with early passage BMECs cells, the expression of senescence-relevant apoptosis-related gene were significantly changed in BME65Cs cells. P16INK4a was downregulated, p53 was low expressed and Bax/Bcl-2 ratio was reversed. Moreover, a slight upregulation of the oncogene c-Myc, along with an undetectable level of breast tumor-related gene Bag-1 and TRPS-1, was observed in BME65Cs cells while these genes are all highly expressed in MCF-7. In addition, DNMT1 is upregulated in BME65Cs. These results suggest that the inhibition of both senescence and mitochondrial apoptosis signalling pathways contribute to the immortality of BME65Cs cells. The expression of p53 and p16INK4a in BME65Cs was altered in the pattern of down-regulation but not "loss", suggesting that this spontaneous immortalization is possibly initiated by other mechanism rather than gene mutation of p53 or p16INK4a. Conclusions Spontaneously immortalised BME65Cs cells maintain many characteristics of normal BMEC cells and

  7. Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells

    Science.gov (United States)

    2013-01-01

    Introduction Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. Methods MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann–Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to

  8. Prepubertal unilateral gynecomastia and the presence of 47,XXY mosaicism in breast epithelial cells: a case report.

    Science.gov (United States)

    Andersen, Peter Stemann; Petersen, Bodil Laub; Juul, Anders; Andersen, Mikael

    2013-02-01

    Breast enlargement in prepubertal boys is a rare condition. This case report describes an otherwise healthy 3-year old non-obese boy who developed a large unilateral cystic breast mass measuring approximately 9 × 6 × 4 cm. The mass was initially treated as a lymphatic malformation, and sclerotherapy with Picibanil (OK-432) was attempted without any detectable effect on size. The mass was later excised. The pathological examination revealed mammary gland tissue suggestive of idiopathic gynecomastia. FISH revealed 47, XXY mosaicism in the abnormal breast epithelial cells, but not in peripheral blood lymphocytes. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. TOX3 is expressed in mammary ER(+) epithelial cells and regulates ER target genes in luminal breast cancer.

    Science.gov (United States)

    Seksenyan, Akop; Kadavallore, Asha; Walts, Ann E; de la Torre, Brian; Berel, Dror; Strom, Samuel P; Aliahmad, Parinaz; Funari, Vincent A; Kaye, Jonathan

    2015-01-30

    A breast cancer susceptibility locus has been mapped to the gene encoding TOX3. Little is known regarding the expression pattern or biological role of TOX3 in breast cancer or in the mammary gland. Here we analyzed TOX3 expression in murine and human mammary glands and in molecular subtypes of breast cancer, and assessed its ability to alter the biology of breast cancer cells. We used a cell sorting strategy, followed by quantitative real-time PCR, to study TOX3 gene expression in the mouse mammary gland. To study the expression of this nuclear protein in human mammary glands and breast tumors, we generated a rabbit monoclonal antibody specific for human TOX3. In vitro studies were performed on MCF7, BT474 and MDA-MB-231 cell lines to study the effects of TOX3 modulation on gene expression in the context of breast cancer cells. We found TOX3 expression in estrogen receptor-positive mammary epithelial cells, including progenitor cells. A subset of breast tumors also highly expresses TOX3, with poor outcome associated with high expression of TOX3 in luminal B breast cancers. We also demonstrate the ability of TOX3 to alter gene expression in MCF7 luminal breast cancer cells, including cancer relevant genes TFF1 and CXCR4. Knockdown of TOX3 in a luminal B breast cancer cell line that highly expresses TOX3 is associated with slower growth. Surprisingly, TOX3 is also shown to regulate TFF1 in an estrogen-independent and tamoxifen-insensitive manner. These results demonstrate that high expression of this protein likely plays a crucial role in breast cancer progression. This is in sharp contrast to previous studies that indicated breast cancer susceptibility is associated with lower expression of TOX3. Together, these results suggest two different roles for TOX3, one in the initiation of breast cancer, potentially related to expression of TOX3 in mammary epithelial cell progenitors, and another role for this nuclear protein in the progression of cancer. In addition, these

  10. Epithelial-Mesenchymal Transition and Breast Cancer

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    Yanyuan Wu

    2016-01-01

    Full Text Available Breast cancer is the most common cancer in women and distant site metastasis is the main cause of death in breast cancer patients. There is increasing evidence supporting the role of epithelial-mesenchymal transition (EMT in tumor cell progression, invasion, and metastasis. During the process of EMT, epithelial cancer cells acquire molecular alternations that facilitate the loss of epithelial features and gain of mesenchymal phenotype. Such transformation promotes cancer cell migration and invasion. Moreover, emerging evidence suggests that EMT is associated with the increased enrichment of cancer stem-like cells (CSCs and these CSCs display mesenchymal characteristics that are resistant to chemotherapy and target therapy. However, the clinical relevance of EMT in human cancer is still under debate. This review will provide an overview of current evidence of EMT from studies using clinical human breast cancer tissues and its associated challenges.

  11. Oscillation of Clock and Clock Controlled Genes Induced by Serum Shock in Human Breast Epithelial and Breast Cancer Cells: Regulation by Melatonin

    Science.gov (United States)

    Xiang, S.; Mao, L.; Duplessis, T.; Yuan, L.; Dauchy, R.; Dauchy, E.; Blask, D.E.; Frasch, T.; Hill, S.M.

    2012-01-01

    This study investigates differences in expression of clock and clock-controlled genes (CCGs) between human breast epithelial and breast cancer cells and breast tumor xenografts in circadian intact rats and examines if the pineal hormone melatonin influences clock gene and CCG expression. Oscillation of clock gene expression was not observed under standard growth conditions in vitro, however, serum shock (50% horse serum for 2 h) induced oscillation of clock gene and CCG expression in MCF-10A cells, which was repressed or disrupted in MCF-7 cells. Melatonin administration following serum shock differentially suppressed or induced clock gene (Bmal1 and Per2) and CCG expression in MCF10A and MCF-7 cells. These studies demonstrate the lack of rhythmic expression of clock genes and CCGs of cells in vitro and that transplantation of breast cancer cells as xenografts into circadian competent hosts re-establishes a circadian rhythm in the peripheral clock genes of tumor cells. PMID:23012497

  12. Sclerotium rolfsii lectin induces stronger inhibition of proliferation in human breast cancer cells than normal human mammary epithelial cells by induction of cell apoptosis.

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    Mohammed Azharuddin Savanur

    Full Text Available Sclerotium rolfsii lectin (SRL isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75, non-tumorigenic breast epithelial cells (MCF-10A and normal mammary epithelial cells (HMECs. SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.

  13. Sclerotium rolfsii lectin induces stronger inhibition of proliferation in human breast cancer cells than normal human mammary epithelial cells by induction of cell apoptosis.

    Science.gov (United States)

    Savanur, Mohammed Azharuddin; Eligar, Sachin M; Pujari, Radha; Chen, Chen; Mahajan, Pravin; Borges, Anita; Shastry, Padma; Ingle, Arvind; Kalraiya, Rajiv D; Swamy, Bale M; Rhodes, Jonathan M; Yu, Lu-Gang; Inamdar, Shashikala R

    2014-01-01

    Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.

  14. Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

    Science.gov (United States)

    Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

    2015-01-01

    The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting. PMID:26068810

  15. Hybrid Cells Derived from Human Breast Cancer Cells and Human Breast Epithelial Cells Exhibit Differential TLR4 and TLR9 Signaling

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    Songül Tosun

    2016-05-01

    Full Text Available TLRs are important receptors of cells of the innate immune system since they recognize various structurally conserved molecular patterns of different pathogens as well as endogenous ligands. In cancer, the role of TLRs is still controversial due to findings that both regression and progression of tumors could depend on TLR signaling. In the present study, M13SV1-EGFP-Neo human breast epithelial cells, MDA-MB-435-Hyg human breast cancer cells and two hybrids M13MDA435-1 and -3 were investigated for TLR4 and TLR9 expression and signaling. RT-PCR data revealed that LPS and CpG-ODN induced the expression of pro-inflammatory cytokines, like IFN-β, TNF-α, IL-1β and IL-6 in hybrid cells, but not parental cells. Interestingly, validation of RT-PCR data by Western blot showed detectable protein levels solely after LPS stimulation, suggesting that regulatory mechanisms are also controlled by TLR signaling. Analysis of pAKT and pERK1/2 levels upon LPS and CpG-ODN stimulation revealed a differential phosphorylation pattern in all cells. Finally, the migratory behavior of the cells was investigated showing that both LPS and CpG-ODN potently blocked the locomotory activity of the hybrid cells in a dose-dependent manner. In summary, hybrid cells exhibit differential TLR4 and TLR9 signaling.

  16. Selective androgen receptor modulators (SARMs negatively regulate triple-negative breast cancer growth and epithelial:mesenchymal stem cell signaling.

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    Ramesh Narayanan

    Full Text Available The androgen receptor (AR is the most highly expressed steroid receptor in breast cancer with 75-95% of estrogen receptor (ER-positive and 40-70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer.Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC co-culture signaling studies were performed to understand the mechanisms of action.Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures.1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.

  17. Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

    Science.gov (United States)

    Dutta, Sujoy; Warshall, Case; Bandyopadhyay, Chirosree; Dutta, Dipanjan; Chandran, Bala

    2014-01-01

    Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment. PMID:24831807

  18. Time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer.

    Science.gov (United States)

    Nakles, Rebecca E; Millman, Sarah L; Cabrera, M Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S; Schroeder, Timm; Furth, Priscilla A

    2013-02-08

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

  19. Downregulation of β-catenin decreases the tumorigenicity, but promotes epithelial-mesenchymal transition in breast cancer cells

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    Kai Cai

    2014-01-01

    Full Text Available Background: Wnt/β-catenin signaling pathway plays a key role in human breast cancer progression. In this study, we down regulated β-catenin expression in human breast cancer MDA-MB-231 cells and investigated the effect of β-catenin knockdown on the cell biological characteristics. Materials and Methods: The recombinant plasmids of pSUPER-enhancement green fluorescent protein 1 (EGFP1-scrabble-β-catenin-short hairpin ribonucleic acid (shRNA and pSUPER-EGFP1-β-catenin-shRNA-1 were transfected into MDA-MB-231 cells, respectively, and the stably transfected cells were isolated from G418 selected clones. The β-catenin gene silenced efficiency was measured by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR and Western blot. The biological characteristics of MDA-MB-231 cells with down regulated β-catenin were evaluated by analyzing cell proliferation, clonogenicity, cell mobility and tumorigenicity. The expression of E-cadherin and Vimentin was concurrently detected by QRT-PCR. Results: The β-catenin-shRNA-1 stably transfected MDA-MB-231 cells significantly decreased β-catenin expression, cell proliferation, clonogenicity, and tumorigenicity in Balb/c nude mice compared with the MDA-MB-231 cells transfected with pSUPER-EGFP1-scrabble-β-catenin-shRNA. Interestingly, knockdown of β-catenin led to the reduction of epithelial E-cadherin expression, the increase of cell mobility and mesenchymal vimentin expression in MDA-MB-231 cells, indicating an epithelial to mesenchymal transition. Conclusion: Knockdown of β-catenin expression in human breast cancer MDA-MB-231 cells inhibits cell tumorigenicity in mice, but promotes cell epithelial-mesenchymal transition.

  20. Deficiency of CCN5/WISP-2-Driven Program in breast cancer Promotes Cancer Epithelial cells to mesenchymal stem cells and Breast Cancer growth.

    Science.gov (United States)

    Das, Amlan; Dhar, Kakali; Maity, Gargi; Sarkar, Sandipto; Ghosh, Arnab; Haque, Inamul; Dhar, Gopal; Banerjee, Snigdha; Banerjee, Sushanta K

    2017-04-27

    Breast cancer progression and relapse is conceivably due to tumor initiating cells (TICs)/cancer stem cells. EMT (epithelial-mesenchymal-transition)-signaling regulates TICs' turnover. However, the mechanisms associated with this episode are unclear. We show that, in triple-negative-breast cancer (TNBC) cells enriched with TICs, CCN5 significantly blocks cellular growth via apoptosis, reversing EMT-signaling and impairing mammosphere formation, thereby blocking the tumor-forming ability and invasive capacity of these cells. To corroborate these findings, we isolated tumor-initiating side populations (SP) and non-side population (NSP or main population) from MCF-7 cell line, and evaluated the impact of CCN5 on these subpopulations. CCN5 was overexpressed in the NSP but downregulated in the SP. Characteristically, NSP cells are ER-α positive and epithelial type with little tumorigenic potency, while SP cells are very similar to triple-negative ones that do not express ER-α- and Her-2 and are highly tumorigenic in xenograft models. The overexpression of CCN5 in SP results in EMT reversion, ER-α upregulation and delays in tumor growth in xenograft models. We reasoned that CCN5 distinguishes SP and NSP and could reprogram SP to NSP transition, thereby delaying tumor growth in the xenograft model. Collectively, we reveal how CCN5-signaling underlies the driving force to prevent TNBC growth and progression.

  1. Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.

    Science.gov (United States)

    Gelfand, Robert; Vernet, Dolores; Bruhn, Kevin; Vadgama, Jaydutt; Gonzalez-Cadavid, Nestor F

    2016-06-01

    Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0-2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.

  2. Identification of H-Ras-Specific Motif for the Activation of Invasive Signaling Program in Human Breast Epithelial Cells

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    Hae-Young Yong

    2011-02-01

    Full Text Available Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR, consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

  3. Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition

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    Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Villadsen, Rene; Rank, Fritz; Bissell, Mina J.; Petersen, Ole William

    2001-10-04

    The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and {beta}4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal. Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce {alpha}-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumorassociated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be

  4. Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition.

    Science.gov (United States)

    Gudjonsson, Thorarinn; Rønnov-Jessen, Lone; Villadsen, René; Rank, Fritz; Bissell, Mina J; Petersen, Ole William

    2002-01-01

    The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and beta4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal. Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce alpha-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumor-associated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be recapitulated

  5. PAC down-regulates estrogen receptor alpha and suppresses epithelial-to-mesenchymal transition in breast cancer cells.

    Science.gov (United States)

    Al-Howail, Huda A; Hakami, Hana A; Al-Otaibi, Basem; Al-Mazrou, Amer; Daghestani, Maha H; Al-Jammaz, Ibrahim; Al-Khalaf, Huda H; Aboussekhra, Abdelilah

    2016-07-27

    Triple-negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and very poor prognosis following progression after standard chemotherapeutic regimens. Therefore, novel molecules and therapeutic options are urgently needed for this category of patients. Recently, we have identified PAC as a curcumin analogue with potent anti-cancer features. HPLC was used to evaluate the stability of PAC and curcumin in PBS and also in circulating blood. Cytotoxicity/apoptosis was assessed in different breast cancer cell lines using propidium iodide/annexinV associated with flow cytometry. Furthermore, immunoblotting analysis determined the effects of PAC on different oncogenic proteins and pathways. Additionally, the real time xCELLigence RTCA technology was applied to investigate the effect of PAC on the cellular proliferation, migration and invasion capacities. PAC is more stable than curcumin in PBS and in circulating blood. Furthermore, we have shown differential sensitivity of estrogen receptor-alfa positive (ERα(+)) and estrogen receptor alfa negative (ERα(-)) breast cancer cells to PAC, which down-regulated ERα in both cell types. This led to complete disappearance of ERα in ERα(-) cells, which express very low level of this receptor. Interestingly, specific down-regulation of ERα in receptor positive cells increased the apoptotic response of these cells to PAC, confirming that ERα inhibits PAC-dependent induction of apoptosis, which could be mediated through ERα down-regulation. Additionally, PAC inhibited the proliferation and suppressed the epithelial-to-mesenchymal transition process in breast cancer cells, with higher efficiency on the TNBC subtype. This effect was also observed in vivo on tumor xenografts. Additionally, PAC suppressed the expression/secretion of 2 important cytokines IL-6 and MCP-1, and consequently inhibited the paracrine procarcinogenic effects of breast cancer cells on breast stromal fibroblasts

  6. A molecular signature of normal breast epithelial and stromal cells from Li-Fraumeni syndrome mutation carriers.

    Science.gov (United States)

    Herbert, Brittney-Shea; Chanoux, Rebecca A; Liu, Yunlong; Baenziger, Peter H; Goswami, Chirayu P; McClintick, Jeanette N; Edenberg, Howard J; Pennington, Robert E; Lipkin, Steven M; Kopelovich, Levy

    2010-10-01

    Specific changes in gene expression during cancer initiation should enable discovery of biomarkers for risk assessment, early detection and targets for chemoprevention. It has been previously demonstrated that altered mRNA and proteome signatures of morphologically normal cells bearing a single inherited "hit" in a tumor suppressor gene parallel many changes observed in the corresponding sporadic cancer. Here, we report on the global gene expression profile of morphologically normal, cultured primary breast epithelial and stromal cells from Li-Fraumeni syndrome (LFS) TP53 mutation carriers. Our analyses identified multiple changes in gene expression in both morphologically normal breast epithelial and stromal cells associated with TP53 haploinsufficiency, as well as interlocking pathways. Notably, a dysregulated p53 signaling pathway was readily detectable. Pharmacological intervention with the p53 rescue compounds CP-31398 and PRIMA-1 provided further evidence in support of the central role of p53 in affecting these changes in LFS cells and treatment for this cancer. Because loss of signaling mediated by TP53 is associated with the development and survival of many human tumors, identification of gene expression profiles in morphologically normal cells that carry "one-hit" p53 mutations may reveal novel biomarkers, enabling the discovery of potential targets for chemoprevention of sporadic tumors as well.

  7. Parabens can enable hallmarks and characteristics of cancer in human breast epithelial cells: a review of the literature with reference to new exposure data and regulatory status.

    Science.gov (United States)

    Darbre, Philippa D; Harvey, Philip W

    2014-09-01

    A framework for understanding the complexity of cancer development was established by Hanahan and Weinberg in their definition of the hallmarks of cancer. In this review, we consider the evidence that parabens can enable development in human breast epithelial cells of four of six of the basic hallmarks, one of two of the emerging hallmarks and one of two of the enabling characteristics. In Hallmark 1, parabens have been measured as present in 99% of human breast tissue samples, possess oestrogenic activity and can stimulate sustained proliferation of human breast cancer cells at concentrations measurable in the breast. In Hallmark 2, parabens can inhibit the suppression of breast cancer cell growth by hydroxytamoxifen, and through binding to the oestrogen-related receptor gamma may prevent its deactivation by growth inhibitors. In Hallmark 3, in the 10 nm-1 μm range, parabens give a dose-dependent evasion of apoptosis in high-risk donor breast epithelial cells. In Hallmark 4, long-term exposure (>20 weeks) to parabens leads to increased migratory and invasive activity in human breast cancer cells, properties that are linked to the metastatic process. As an emerging hallmark methylparaben has been shown in human breast epithelial cells to increase mTOR, a key regulator of energy metabolism. As an enabling characteristic parabens can cause DNA damage at high concentrations in the short term but more work is needed to investigate long-term, low-dose mixtures. The ability of parabens to enable multiple cancer hallmarks in human breast epithelial cells provides grounds for regulatory review of the implications of the presence of parabens in human breast tissue. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Regulation of matrix stiffness on the epithelial-mesenchymal transition of breast cancer cells under hypoxia environment

    Science.gov (United States)

    Lv, Yonggang; Chen, Can; Zhao, Boyuan; Zhang, Xiaomei

    2017-06-01

    Substrate stiffness and hypoxia are associated with tumor development and progression, respectively. However, the synergy of them on the biological behavior of human breast cancer cell is still largely unknown. This study explored how substrate stiffness regulates the cell phenotype, viability, and epithelial-mesenchymal transition (EMT) of human breast cancer cells MCF-7 under hypoxia (1% O2). TRITC-phalloidin staining showed that MCF-7 cells transformed from round to irregular polygon with stiffness increase either in normoxia or hypoxia. While being accompanied with the upward tendency from a 0.5- to a 20-kPa substrate, the percentage of cell apoptosis was significantly higher in hypoxia than that in normoxia, especially on the 20-kPa substrate. Additionally, it was hypoxia, but not normoxia, that promoted the EMT of MCF-7 by upregulating hypoxia-inducible factor-1α (HIF-1α), vimentin, Snail 1, and matrix metalloproteinase 2 (MMP 2) and 9 (MMP 9), and downregulating E-cadherin simultaneously regardless of the change of substrate stiffness. In summary, this study discovered that hypoxia and stiffer substrate (20 kPa) could synergistically induce phenotype change, apoptosis, and EMT of MCF-7 cells. Results of this study have an important significance on further exploring the synergistic effect of stiffness and hypoxia on the EMT of breast cancer cells and its molecular mechanism.

  9. SHOX2 Is a Direct miR-375 Target and a Novel Epithelial-to-Mesenchymal Transition Inducer in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sungguan Hong

    2014-04-01

    Full Text Available MicroRNAs have added a new dimension to our understanding of tumorigenesis and associated processes like epithelial-to-mesenchymal transition (EMT. Here, we show that miR-375 is elevated in epithelial-like breast cancer cells, and ectopic miR-375 expression suppresses EMT in mesenchymal-like breast cancer cells. We identified short stature homeobox 2 (SHOX2 as a miR-375 target, and miR-375–mediated suppression in EMT was reversed by forced SHOX2 expression. Ectopic SHOX2 expression can induce EMT in epithelial-like breast cancer cells, whereas SHOX2 knockdown diminishes EMT traits in mesenchymal-like breast cancer cells, demonstrating SHOX2 as an EMT inducer. We show that SHOX2 acts as a transcription factor to upregulate transforming growth factor β receptor I (TβR-I expression, and TβR-I inhibitor LY364947 abolishes EMT elicited by ectopic SHOX2 expression, suggesting that transforming growth factor β signaling is essential for SHOX2-induced EMT. Manipulating SHOX2 abundance in breast cancer cells impact in vitro invasion and in vivo dissemination. Analysis of breast tumor microarray database revealed that high SHOX2 expression significantly correlates with poor patient survival. Our study supports a critical role of SHOX2 in breast tumorigenicity.

  10. Oct-4 and Nanog promote the epithelial-mesenchymal transition of breast cancer stem cells and are associated with poor prognosis in breast cancer patients

    Science.gov (United States)

    Luo, Minna; Zhang, Xin; Wei, Xiaofei; Gao, Jiyue; Zhao, Zuowei; Liu, Caigang

    2014-01-01

    Oct-4 and Nanog in regulating the epithelial-mesenchymal transition (EMT) and metastasis of breast cancer has not been clarified. We found that both Oct-4 and Nanog expression were significantly associated with tumor pathology and poor prognosis in 126 breast cancer patients. Characterization of CD44+CD24-Cancer stem cell(CSC) derived from breast cancer cells indicated that CSC rapidly formed mammospheres and had potent tumorigenicity in vivo. Furthermore, TGF-β up-regulated the expression of Oct-4, Nanog, N-cadherin, vimentin, Slug, and Snail, but down-regulated E-cadherin and cytokeratin 18 expression, demonstrating that CSC underwent EMT. Knockdown of both Oct-4 and Nanog expression inhibited spontaneous changes in the expression of EMT-related genes, while induction of both Oct-4 and Nanog over-expression enhanced spontaneous changes in the expression of EMT-related genes in CSC. However, perturbing alternation of Oct-4 and Nanog expression also modulated TGF-β-induced EMT-related gene expression in CSC. Induction of Oct-4 and Nanog over-expression enhanced the invasiveness of CSC, but knockdown of both Oct-4 and Nanog inhibited the migration of CSC in vitro. Our data suggest that both Oct-4 and Nanog may serve as biomarkers for evaluating breast cancer prognosis. Our findings indicate that Oct-4 and Nanog positively regulate the EMT process, contributing to breast cancer metastasis. PMID:25301732

  11. Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Qingbei Zhang

    Full Text Available Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3 and basal (MDA-MB-231 types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL and glioblastoma cell lines (U87 and D54. These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.

  12. Effects of bisphenol compounds on the growth and epithelial mesenchymal transition of MCF-7 CV human breast cancer cells

    Science.gov (United States)

    Kim, Ji-Youn; Choi, Ho-Gyu; Lee, Hae-Miru; Lee, Geum-A; Hwang, Kyung-A; Choi, Kyung-Chul

    2017-01-01

    Bisphenol-A (BPA) has been considered as an endocrine disrupting chemical (EDC) because it can exert estrogenic properties. For bisphenol-S (BPS) and bisphenol-F (BPF) that are BPA analogs and substitutes, their risk to estrogen-dependent cancer has been reported rarely compared with the numerous cases of BPA. In this study, we examined whether BPA, BPS, and BPF can lead to the proliferation, migration, and epithelial mesenchymal transition (EMT) of MCF-7 clonal variant (MCF-7 CV) breast cancer cells expressing estrogen receptors (ERs). In a cell viability assay, BPA, BPS, and BPF significantly increased proliferation of MCF-7 CV cells compared to control (DMSO) as did 17β-estradiol (E2). In Western blotting assay, BPA, BPS, and BPF enhanced the protein expression of cell cycle progression genes such as cyclin D1 and E1. In addition, MCF-7 CV cells lost cell to cell contacts and acquired fibroblast-like morphology by the treatment of BPA, BPS, or BPF for 24 hours. In cell migration assay, BPA, BPS, and BPF accelerated the migration capability of MCF-7 CV cells as did E2. In relation with the EMT process, BPA, BPS, and BPF increased the protein expression ofN-cadherin, while they decreased the protein expression of E-cadherin. When BPA, BPS, and BPF were co-treated with ICI 182,780, an ER antagonist, proliferation effects were reversed, the expression of cyclin D1 and cyclin E1 was downregulated, and the altered cell migration and expression ofN-cadherin and E-cadherin by BPA, BPS, and BPF were restored to the control level. Thus, these results imply that BPS and BPF also have the risk of breast cancer progression as much as BPA in the induction of proliferation and migration of MCF-7 CV cells by regulating the protein expression of cell cycle-related genes and EMT markersvia the ER-dependent pathway. PMID:28808208

  13. Apoptotic ratios and mitotic abnormalities in 17-β-estradiol-transformed human breast epithelial MCF-10F cells

    Directory of Open Access Journals (Sweden)

    LMS Cruz

    Full Text Available Treatment of human breast epithelial cells MCF-10F with 17-β-estradiol has been reported to result in E2-transformed cells which have given rise to highly invasive C5 cells that in turn generate tumors in SCID mice. From these tumors, various cell lines, among which C5-A6-T6 and C5-A8-T8, were obtained. Although different phases of the tumorigenesis process in this model have been studied in molecular biology and image analysis assays, no cytological data on apoptotic ratios and mitotic abnormalities have been established to accompany the various steps leading to 17-β-estradiol-treated MCF-10F cells to tumorigenesis. Here we detected that the apoptotic ratio decreases with the transformation and tumorigenesis progress, except for the tumor cell line C5-A8-T8, probably on account of its more intense proliferation rate and a more rapid culture medium consumption. Increased frequency of mitotic abnormalities contributed by triple- and tetrapolar metaphases, and by lagging chromosomes and chromosome bridges observed at the anaphase found by transformation and tumorigenesis progress. However, no difference was found under these terms when the C5-A6-T6 and C5-A8-T8 tumor cell lines were compared to each other. Present findings are in agreement with the nuclear instability and enrichment of dysregulated genes in the apoptotic process promoted by transformation and tumorigenesis in 17-β-estradiol-treated MCF-10F cells.

  14. Inhibition of Epithelial-mesenchymal Transition in Response to Treatment with Metformin and Y27632 in Breast Cancer Cell Lines.

    Science.gov (United States)

    Leonel, Camila; Ferreira, Lívia Carvalho; Borin, Thaiz Ferraz; Moschetta, Marina Gobbe; Freitas, Gabriela Scavacini; Haddad, Michel Raineri; de Camargos Pinto Robles, João Antonio; Aparecida Pires de Campos Zuccari, Debora

    2017-01-01

    ROCK-1 expression is associated with the malignant character of tumors, while inhibiting this molecule results in a significant suppression of tumor metastasis. Likewise, transforming growth factor beta (TGF-β) is associated with this malignancy by having the ability to induce epithelial-mesenchymal transition (EMT). Metformin, a drug used in the treatment of diabetes, has previously been shown to inhibit EMT in breast cancer cells. The aim of this study is to evaluate the TGF-β1 action model for induction of EMT and the action of metformin and ROCK-1 inhibitor (Y27632) in EMT process in breast cancer cell lines. MCF-7 and MDA-MB-231 cell lines were treated with metformin and Y27632, after induction of EMT by TGF-β1, to examine the effects on cell migration as well as the protein expression of the ROCK-1 markers, vimentin, E-cadherin, CD44 and CD24 by immunocitochemistry. There was a lower protein expression of ROCK-1, vimentin, CD44 and CD24 in both cell lines after treatment with metformin and Y27632. In MDA-MB-231 cells, E-cadherin expression was increased in all treatment groups. Treatment of MDA-MB-231 cell line with metformin and Y27632 significantly reduced the invasion of these cells. This study confirms the benefits of metformin and Y27632 as potential therapeutic agents in mammary tumors, by blocking EMT process and metastatic potential. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Anti-Cancer Activity of Solanum nigrum (AESN through Suppression of Mitochondrial Function and Epithelial-Mesenchymal Transition (EMT in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ying-Jang Lai

    2016-04-01

    Full Text Available Chemotherapy is the main approach for treating advanced and recurrent carcinoma, but the clinical performance of chemotherapy is limited by relatively low response rates, drug resistance, and adverse effects that severely affect the quality of life of patients. An association between epithelial-mesenchymal transition (EMT and chemotherapy resistance has been investigated in recent studies. Our recent studies have found that the aqueous extract of Solanum nigrum (AESN is a crucial ingredient in some traditional Chinese medicine formulas for treating various types of cancer patients and exhibits antitumor effects. We evaluated the suppression of EMT in MCF-7 breast cancer cells treated with AESN. The mitochondrial morphology was investigated using Mitotracker Deep-Red FM stain. Our results indicated that AESN markedly inhibited cell viability of MCF-7 breast cancer cells through apoptosis induction and cell cycle arrest mediated by activation of caspase-3 and production of reactive oxygen species. Furthermore, mitochondrial fission was observed in MCF-7 breast cancer cells treated with AESN. In addition to elevation of E-cadherin, downregulations of ZEB1, N-cadherin, and vimentin were found in AESN-treated MCF-7 breast cancer cells. These results suggested that AESN could inhibit EMT of MCF-7 breast cancer cells mediated by attenuation of mitochondrial function. AESN could be potentially beneficial in treating breast cancer cells, and may be of interest for future studies in developing integrative cancer therapy against proliferation, metastasis, and migration of breast cancer cells.

  16. BRCA1 requirement for the fidelity of plasmid DNA double-strand break repair in cultured breast epithelial cells.

    Science.gov (United States)

    Thompson, Eric G; Fares, Hanna; Dixon, Kathleen

    2012-01-01

    The tumor suppressor breast cancer susceptibility protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks (DSBs). BRCA1 promotes the error-free repair of DSBs through homologous recombination and is also implicated in the regulation of nonhomologous end joining (NHEJ) repair fidelity. Here, we investigate the role of BRCA1 in NHEJ repair mutagenesis following a DSB. We examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of DSB repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of meiotic recombination enzyme 11 (Mre11) 3'-5'-exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA mutagenesis and MMEJ following a DSB. Inhibition of Mre11-exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid DSB repair. The results suggest that BRCA1 protects DNA from mutagenesis during nonhomologous DSB repair in plasmid-based assays. The increased frequency of DSB mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis. Copyright © 2011 Wiley Periodicals, Inc.

  17. Aspirin blocks growth of breast tumor cells and tumor-initiating cells and induces reprogramming factors of mesenchymal to epithelial transition.

    Science.gov (United States)

    Maity, Gargi; De, Archana; Das, Amlan; Banerjee, Snigdha; Sarkar, Sandipto; Banerjee, Sushanta K

    2015-07-01

    Acetylsalicylic acid (ASA), also known as aspirin, a classic, nonsteroidal, anti-inflammatory drug (NSAID), is widely used to relieve minor aches and pains and to reduce fever. Epidemiological studies and other experimental studies suggest that ASA use reduces the risk of different cancers including breast cancer (BC) and may be used as a chemopreventive agent against BC and other cancers. These studies have raised the tempting possibility that ASA could serve as a preventive medicine for BC. However, lack of in-depth knowledge of the mechanism of action of ASA reshapes the debate of risk and benefit of using ASA in prevention of BC. Our studies, using in vitro and in vivo tumor xenograft models, show a strong beneficial effect of ASA in the prevention of breast carcinogenesis. We find that ASA not only prevents breast tumor cell growth in vitro and tumor growth in nude mice xenograft model through the induction of apoptosis, but also significantly reduces the self-renewal capacity and growth of breast tumor-initiating cells (BTICs)/breast cancer stem cells (BCSCs) and delays the formation of a palpable tumor. Moreover, ASA regulates other pathophysiological events in breast carcinogenesis, such as reprogramming the mesenchymal to epithelial transition (MET) and delaying in vitro migration in BC cells. The tumor growth-inhibitory and reprogramming roles of ASA could be mediated through inhibition of TGF-β/SMAD4 signaling pathway that is associated with growth, motility, invasion, and metastasis in advanced BCs. Collectively, ASA has a therapeutic or preventive potential by attacking possible target such as TGF-β in breast carcinogenesis.

  18. Pregnancy at early age is associated with a reduction of progesterone-responsive cells and epithelial Wnt signaling in human breast tissue.

    Science.gov (United States)

    Muenst, Simone; Mechera, Robert; Däster, Silvio; Piscuoglio, Salvatore; Ng, Charlotte K Y; Meier-Abt, Fabienne; Weber, Walter P; Soysal, Savas D

    2017-04-04

    Pregnancy at early age is the most significant modifiable factor which consistently decreases lifetime breast cancer risk. However, the underlying mechanisms haven't been conclusively identified. Studies in mice suggest a reduction in progesterone-receptor (PR) sensitive epithelial cells as well as a downregulation of the Wnt signaling pathway as being one of the main mechanisms for the protective effect of early pregnancy. The aim of our study was to validate these findings in humans. We collected benign breast tissue of 125 women who had been stratified according to age at first pregnancy and the occurrence of subsequent breast cancer, and performed immunohistochemistry for PR, Wnt4 and the Wnt-target Versican. The number of PR positive epithelial cells was significantly lower in the group of women with early pregnancy and no subsequent breast cancer compared to the group of nulliparous women with subsequent invasive breast cancer (p = 0.0135). In women with early pregnancy, expression of Versican and Wnt4 was significantly lower compared to nulliparous women (p = 0.0036 and p = 0.0241 respectively), and Versican expression was also significant lower compared to women with late pregnancy (p pregnancy in humans. This results in a decreased proliferation of stem/progenitor cells; therefore, the Wnt signaling pathway may represent a potential target for breast cancer prevention in humans.

  19. Continuous in vitro exposure to low dose genistein induces genomic instability in breast epithelial cells

    Science.gov (United States)

    Kim, Young Mi; Yang, Shihe; Xu, Weihong; Li, Shibo; Yang, Xiaohe

    2008-01-01

    Genistein is a major soy isoflavone with multiple properties. The impact of soy/genistein on breast cancer is controversial. One of the issues is whether soy/genistein has a genotoxic effect at physiological concentrations. To address this question using an in vitro model, we first established MCF-10A/G0 and MCF-10A/G1 cell lines, which were MCF-10A cells exposed to 0.01% DMSO (as vehicle control), i.e. MCF-10A/G0, or 1 µM of genistein for three months, MCF-10A/G1, respectively. Chromosomal changes were compared between the two cell lines by routine G-banded chromosome analyses, regular CGH and oligo array-based CGH. After three months of exposure to genistein, the cell line MCF-10A/G1 showed a loss of a normal chromosome 8, a gain of extra chromosome 20, plus a loss of a chromosomal segment on the short arm of chromosome 9, which leads to a homozygous deletion of the tumor suppressor genes, INK4/p16 and INK4/p15. Our results suggest that long-term/low concentration exposure to genistein may have the potential to induce chromosomal imbalances. These genotoxic effects may work in concert with other factors to induce genetic lesions that contribute to soy/genistein associated risk. PMID:18940470

  20. Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

    Directory of Open Access Journals (Sweden)

    Marion Chapellier

    2015-02-01

    Full Text Available Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

  1. Elucidation of epithelial-mesenchymal transition-related pathways in a triple-negative breast cancer cell line model by multi-omics interactome analysis

    DEFF Research Database (Denmark)

    Pauling, Josch K; Christensen, Anne G; Batra, Richa

    2014-01-01

    gene expression, protein expression or post-translational modifications. To overcome single omics analysis, we developed a set of computational methods that allow a combined analysis of data collections from multiple omics fields utilizing hybrid interactome networks. We apply these methods to data...... obtained from a triple-negative breast cancer cell line model, combining data sets of gene and protein expression as well as protein phosphorylation. We focus on alterations associated with the phenotypical differences arising from epithelial-mesenchymal transition in two breast cancer cell lines...

  2. Human breast cancer cells are redirected to mammary epithelial cells upon interaction with the regenerating mammary gland microenvironment in-vivo.

    Directory of Open Access Journals (Sweden)

    Karen M Bussard

    Full Text Available Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display 'normal' behavior when placed into 'normal' ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for 'normal' gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo.

  3. WT1 expression in breast cancer disrupts the epithelial/mesenchymal balance of tumour cells and correlates with the metabolic response to docetaxel

    Science.gov (United States)

    Artibani, Mara; Sims, Andrew H.; Slight, Joan; Aitken, Stuart; Thornburn, Anna; Muir, Morwenna; Brunton, Valerie G.; Del-Pozo, Jorge; Morrison, Linda R.; Katz, Elad; Hastie, Nicholas D.; Hohenstein, Peter

    2017-01-01

    WT1 is a transcription factor which regulates the epithelial-mesenchymal balance during embryonic development and, if mutated, can lead to the formation of Wilms’ tumour, the most common paediatric kidney cancer. Its expression has also been reported in several adult tumour types, including breast cancer, and usually correlates with poor outcome. However, published data is inconsistent and the role of WT1 in this malignancy remains unclear. Here we provide a complete study of WT1 expression across different breast cancer subtypes as well as isoform specific expression analysis. Using in vitro cell lines, clinical samples and publicly available gene expression datasets, we demonstrate that WT1 plays a role in regulating the epithelial-mesenchymal balance of breast cancer cells and that WT1-expressing tumours are mainly associated with a mesenchymal phenotype. WT1 gene expression also correlates with CYP3A4 levels and is associated with poorer response to taxane treatment. Our work is the first to demonstrate that the known association between WT1 expression in breast cancer and poor prognosis is potentially due to cancer-related epithelial-to-mesenchymal transition (EMT) and poor chemotherapy response. PMID:28345629

  4. KiSS1-induced GPR54 signaling inhibits breast cancer cell migration and epithelial-mesenchymal transition via protein kinase D1.

    Science.gov (United States)

    Tan, K; Cho, S-G; Luo, W; Yi, T; Wu, X; Siwko, S; Liu, M; Yuan, W

    2014-01-01

    The metastasis suppressor protein Kisspeptin regulates cancer cell proliferation and motility through its receptor, GRP54. However, the critical downstream effectors remain unclear. In this study, we investigated GPR54 signaling in breast cancer cells. Kisspeptin stimulation caused a decrease in migration of multiple breast cancer cell lines. Also, Kisspeptin inhibited MDA-MB-231 cell colony formation in 3D matrigel culture and in soft agar. Kisspeptin treatment elevated phosphorylated PKD1 in a PKC-dependent manner. However, knockdown of either GPR54 or PKD1 increased breast cancer cell migration and invasion. Furthermore, GPR54 knockdown blocked Kisspeptin-induced phosphorylation of PKD1. Finally, Kisspeptin stimulation induced a PKD1 phosphorylation-dependent decrease in expression of Slug, a transcription factor that drives epithelial-mesenchymal transition (EMT), and a concomitant increase in E-cadherin expression. Therefore, KiSS1/GPR54 signaling through PKD1 acts to maintain the epithelial state and to inhibit breast cancer cell invasiveness, and exerts functions associated with its role as a metastasis suppressor.

  5. E3 Ubiquitin Ligase Cbl-b Prevents Tumor Metastasis by Maintaining the Epithelial Phenotype in Multiple Drug-Resistant Gastric and Breast Cancer Cells.

    Science.gov (United States)

    Xu, Ling; Zhang, Ye; Qu, Xiujuan; Che, Xiaofang; Guo, Tianshu; Cai, Ying; Li, Aodi; Li, Danni; Li, Ce; Wen, Ti; Fan, Yibo; Hou, Kezuo; Ma, Yanju; Hu, Xuejun; Liu, Yunpeng

    2017-04-01

    Multiple drug resistance (MDR) and metastasis are two major factors that contribute to the failure of cancer treatment. However, the relationship between MDR and metastasis has not been characterized. Additionally, the role of the E3 ubiquitin ligase Cbl-b in metastasis of MDR gastric and breast cancer is not well known. In the present study, we found that MDR gastric and breast cancer cells possess a typical mesenchymal phenotype and enhanced cell migration capacity. Additionally, Cbl-b is poorly expressed in MDR gastric and breast cancer cells. In MDR gastric adenocarcinoma tissues, gastric cancer patients with low Cbl-b expression were more likely to have tumor invasion (P=.016) and lymph node metastasis (P=.007). Moreover, overexpression of Cbl-b reduced cell migration in MDR cell cultures both in vitro and in vivo. Cbl-b overexpression also prevented EMT by inducing ubiquitination and degradation of EGFR, leading to inhibition of the EGFR-ERK/Akt-miR-200c-ZEB1 axis. However, further overexpression of EGFR on a background of Cbl-b overexpression restored both the mesenchymal phenotype and cell migration capacity of MDR gastric and breast cancer cells. These results suggest that Cbl-b is an important factor for maintenance of the epithelial phenotype and inhibition of cell migration in MDR gastric and breast cancer cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. The tetraindole SK228 reverses the epithelial-to-mesenchymal transition of breast cancer cells by up-regulating members of the miR-200 family.

    Directory of Open Access Journals (Sweden)

    Chie-Hong Wang

    Full Text Available The results of recent studies have shown that metastasis, the most common malignancy and primary cause of mortality promoted by breast cancer in women, is associated with the epithelial-to-mesenchymal transition (EMT. The results of the current study show that SK228, a novel indole containing substance, exhibits anti-cancer activity. In addition, the effects of SK228 on the regulation of EMT in breast cancer cells as well as the underlying mechanism have been explored. SK228 was observed to induce a fibroblastoid to epithelial-like change in the appearance of various breast cancer cell lines and to suppress the migration and invasion of these cancer cells in vitro. Moreover, expression of E-cadherin was found to increase following SK228 treatment whereas ZEB1 expression was repressed. Expression of other major EMT inducers, including ZEB2, Slug and Twist1, is also repressed by SK228 as a consequence of up-regulation of members of the miR-200 family, especially miR-200c. The results of animal studies demonstrate that SK228 treatment leads to effective suppression of breast cancer growth and metastasis in vivo. The observations made in this investigation show that SK228 reverses the EMT process in breast cancer cells via an effect on the miR-200c/ZEB1/E-cadherin signalling pathway. In addition, the results of a detailed analysis of the in vivo anti-cancer activities of SK228, carried out using a breast cancer xenograft animal model, show that this substance is a potential chemotherapeutic agent for the treatment of breast cancer.

  7. Linc-ROR induces epithelial-mesenchymal transition and contributes to drug resistance and invasion of breast cancer cells.

    Science.gov (United States)

    Chen, Yao-Min; Liu, Yu; Wei, Hai-Yan; Lv, Ke-Zhen; Fu, Peifen

    2016-08-01

    We aimed to investigate the role of large intergenic noncoding RNA regulator of reprogramming (linc-ROR) in the chemotherapy resistance of human breast cancer (BC) cells and its mechanism. A total of 142 patients diagnosed with BC in the First Affiliated Hospital, Zhejiang University between January 2012 and January 2014 were enrolled in our study. The BC tissues and the adjacent normal tissues (5 cm away from tumor tissue) of the enrolled patients were selected, and human BC cell lines (MCF10A, SK-BR-3, MCF-7, Bcap-37, MDA-MB-231, and T47D) were also selected. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, and Transwell were applied in our study. Expression level of linc-ROR messenger RNA (mRNA) in BC tissues was clearly higher than that in adjacent normal tissues, and significant difference was found between expression level of linc-ROR mRNA and lymph node metastasis (all P ROR was highly expressed in others BC cell lines compared with that in immortalized mammary epithelial cells (MECs) MCF10A (both P ROR cell presented decreased sensibility of 5-FU and paclitaxel with decreased E-cadherin expression, increased Vimentin, N-cadherin expression, and invasion ability (all P ROR is an important marker for multidrug resistance of BC, and its up-regulation is important for chemotherapy tolerance and invasion of BC.

  8. Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Panshi Zhang

    Full Text Available Treatments for triple-negative breast cancer (TNBC are limited; intermediate-conductance calcium-activated potassium (SK4 channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC and western blotting (WB, increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05. Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05. Further investigation revealed that treatment with epidermal growth factor (EGF/basic fibroblast growth factor (bFGF caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.

  9. WISP-2: a serum-inducible gene differentially expressed in human normal breast epithelial cells and in MCF-7 breast tumor cells.

    Science.gov (United States)

    Zoubine, M N; Banerjee, S; Saxena, N K; Campbell, D R; Banerjee, S K

    2001-03-30

    WISP-2 is a Wnt-1-induced signaling protein identified as a member of CCN growth factor family. A role for this molecule during tumorigenesis is suspected but remains unproven. Here we show that WISP-2 expression was undetectable, or minimally detectable, in nontransformed human mammary epithelial cells, but was overexpressed in MCF-7 cells. Expression of WISP-2 in MCF-7 cells was modulated by serum and correlated with the serum-induced MCF-7 tumor cell proliferation, suggesting that WISP-2 is serum responsive and may be a positive regulator of tumor cell proliferation. Copyright 2001 Academic Press.

  10. Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary Epithelial Cell Differentiation

    Science.gov (United States)

    2006-03-01

    mouse mammary tissue. Proc. Natl. Acad. Sci. 92, 8831-8835 (1995). 12. Folias, A. et al. BRCA1 interacts directly with the Fanconi anemia protein...germline BRCA1 mutations and an adverse outcome following breast cancer. Br J Cancer 89, 1031-1034. Hakem, R., de la Pompa, J. L., Sirard, C., Mo

  11. Radiation Dose-effects on Cell Cycle, Apoptosis, and Marker Expression of Ataxia Telangiectasia-Heterozygous Human Breast Epithelial Cells

    Science.gov (United States)

    Cruz, A.; Bors, K.; Jansen, H.; Richmond, R.

    2003-01-01

    Ataxia-telangiectasia (A-T) is a radiation-sensitive genetic condition. AT-heterozygous human mammary epithelial cells (HMEC) were irradiated using a Cs137 source in order to compare cell cycle, apoptosis, and marker expression responses across 3 radiation doses. No differences in cell cycle and apoptosis were found with any of the radiation doses used (30, 60, and 90 rads) compared with the unirradiated control (0 rad). At the same doses, however, differences were found in marker expression, such as keratin 18 (kl8), keratin 14 (k14), insulin-like growth factor I receptor (IGF-IR), and connexin 43 (cx43). This may indicate that radiation sensitivity in the heterozygous state may be initiated through signal transduction responses.

  12. TGF-β-Dependent Growth Arrest and Cell Migration in Benign and Malignant Breast Epithelial Cells Are Antagonistically Controlled by Rac1 and Rac1b

    Directory of Open Access Journals (Sweden)

    Catharina Melzer

    2017-07-01

    Full Text Available Despite improvements in diagnosis and treatment, breast cancer is still the most common cancer type among non-smoking females. TGF-β can inhibit breast cancer development by inducing cell cycle arrest in both, cancer cells and, as part of a senescence program in normal human mammary epithelial cells (HMEC. Moreover, TGF-β also drives cell migration and invasion, in part through the small GTPases Rac1 and Rac1b. Depletion of Rac1b or Rac1 and Rac1b in MDA-MB-231 or MDA-MB-435s breast cancer cells by RNA interference enhanced or suppressed, respectively, TGF-β1-induced migration/invasion. Rac1b depletion in MDA-MB-231 cells also increased TGF-β-induced p21WAF1 expression and ERK1/2 phosphorylation. Senescent HMEC (P15/P16, when compared to their non-senescent counterparts (P11/P12, presented with dramatically increased migratory activity. These effects were paralleled by elevated expression of genes associated with TGF-β signaling and metastasis, downregulated Rac1b, and upregulated Rac1. Our data suggest that acquisition of a motile phenotype in HMEC resulted from enhanced autocrine TGF-β signaling, invasion/metastasis-associated gene expression, and a shift in the ratio of antimigratory Rac1b to promigratory Rac1. We conclude that although enhanced TGF-β signaling is considered antioncogenic in HMEC by suppressing oncogene-induced transformation, this occurs at the expense of a higher migration and invasion potential.

  13. Gastric Epithelial Stem Cells

    Science.gov (United States)

    MILLS, JASON C.; SHIVDASANI, RAMESH A.

    2013-01-01

    Advances in our understanding of stem cells in the gastrointestinal tract include the identification of molecular markers of stem and early progenitor cells in the small intestine. Although gastric epithelial stem cells have been localized, little is known about their molecular biology. Recent reports describe the use of inducible Cre recombinase activity to indelibly label candidate stem cells and their progeny in the distal stomach, (ie, the antrum and pylorus). No such lineage labeling of epithelial stem cells has been reported in the gastric body (corpus). Among stem cells in the alimentary canal, those of the adult corpus are unique in that they lie close to the lumen and increase proliferation following loss of a single mature progeny lineage, the acid-secreting parietal cell. They are also unique in that they neither depend on Wnt signaling nor express the surface marker Lgr5. Because pathogenesis of gastric adenocarcinoma has been associated with abnormal patterns of gastric differentiation and with chronic tissue injury, there has been much research on the response of stomach epithelial stem cells to inflammation. Chronic inflammation, as induced by infection with Helicobacter pylori, affects differentiation and promotes metaplasias. Several studies have identified cellular and molecular mechanisms in spasmolytic polypeptide–expressing (pseudopyloric) metaplasia. Researchers have also begun to identify signaling pathways and events that take place during embryonic development that eventually establish the adult stem cells to maintain the specific features and functions of the stomach mucosa. We review the cytologic, molecular, functional, and developmental properties of gastric epithelial stem cells. PMID:21144849

  14. Activation of GPER suppresses epithelial mesenchymal transition of triple negative breast cancer cells via NF-κB signals.

    Science.gov (United States)

    Chen, Zhuo-Jia; Wei, Wei; Jiang, Guan-Min; Liu, Hao; Wei, Wei-Dong; Yang, Xiangling; Wu, Ying-Min; Liu, Huanliang; Wong, Chris K C; Du, Jun; Wang, Hong-Sheng

    2016-06-01

    The targeted therapy for triple-negative breast cancer (TNBC) is a great challenge due to our poor understanding on its molecular etiology. In the present study, our clinical data showed that the expression of G-protein coupled estrogen receptor (GPER) is negatively associated with lymph node metastasis, high-grade tumor and fibronectin (FN) expression while positively associated with the favorable outcome in 135 TNBC patients. In our experimental studies, both the in vitro migration and invasion of TNBC cells were inhibited by GPER specific agonist G-1, through the suppression of the epithelial mesenchymal transition (EMT). The G-1 treatment also reduced the phosphorylation, nuclear localization, and transcriptional activities of NF-κB. While over expression of NF-κB attenuated the action of G-1 in suppressing EMT. Our data further illustrated that the phosphorylation of GSK-3β by PI3K/Akt and ERK1/2 mediated, at least partially, the inhibitory effect of G-1 on NF-κB activities. It was further confirmed in a study of MDA-MB-231 tumor xenografts in nude mice. The data showed that G-1 inhibited the in vivo growth and invasive potential of TNBC via suppression of EMT. Our present study demonstrated that an activation of GPER pathway elicits tumor suppressive actions on TNBC, and supports the use of G-1 therapeutics for TNBC metastasis. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. The role of iodine and delta-iodolactone in growth and apoptosis of malignant thyroid epithelial cells and breast cancer cells.

    Science.gov (United States)

    Gärtner, Roland; Rank, Petra; Ander, Birgit

    2010-01-01

    As we previously demonstrated, the inhibitory effect of iodine on thyroid cell growth is mediated by iodolactones, especially 6-iodo-5-hydroxy-eicosatrienoic acid (delta-iodolactone). In this communication we compare the effect of iodide, molecular iodine and delta-iodolactone on growth inhibition and apoptosis on three human thyroid carcinoma cell lines (B-CPAP cells, FTC-133 cells and 8505C cells) as well as on human breast cancer cells (MCF 7). Thyroid carcinoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and MCF 7 cells in Rowswell Park Memorial Institute (RPMI) culture medium, both containing 10% (v/v) Fetal Calf Serum (FCS), until they were confluent. Around 2000 cells were then distributed in 12-well plates and grown for 48 h in either DMEM (thyroid cancer cells) or in RPMI medium (MCF 7 cells) both containing 5% FCS. Thereafter, different concentrations of iodide, iodine or delta-iodolactone were added for 24 h. Growth rate was estimated by cell counting in a Coulter Counter adapted for epithelial cells. Apoptosis was determined by a mitochondrial potential assay. The growth rate of B-CPAP cells was unaffected by iodide, but was reduced by high concentreations of molecular iodine (100 and 500 microM). However, delta-iodolactone significantly reduced cell proliferation already with low concentrations (5 microM and 10 microM) and further in a dose-dependent manner up to 82%. FTC-133 and 8505C cells were unaffected by iodide, iodine or delta-iodolactone. In contrast, in MCF 7 cells, molecular iodine (100 microM) inhibited growth from 100% to 83% but delta-iodolactone (1, 5 and 10 microM) dose-dependently decreased growth rate from 100% to 82% and 62%, respectively. The inhibition of growth was through apoptosis, and not necrosis, as the amount of apoptotic cells corresponded to the growth inhibition. delta-Iotaodolactone seems to be the main iodocompound which can inhibit growth and induce apoptosis in B-CPAP cells as well as in MCF 7

  16. The potential role of Brachyury in inducing epithelial-to-mesenchymal transition (EMT) and HIF-1α expression in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Chao [Department of Mammary Surgery, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China); Zhang, Jingjing, E-mail: jingjingzhangzs@163.com [Department of Cancer Radiotherapy, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China); Fu, Jianhua [Department of Thoracic Surgery, Cancer Center, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China); Ling, Feihai, E-mail: feihailingfhl@163.com [Department of Mammary Surgery, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China)

    2015-11-27

    One of transcription factors of the T-box family, Brachyury has been implicated in tumorigenesis of many types of cancers, regulating cancer cell proliferation, metastasis, invasion and epithelial-to-mesenchymal transition (EMT). However, the role of Brachyury in breast cancer cells has been scarcely reported. The present study aimed to investigate the expression and role of Brachyury in breast cancer. Brachyury expression was analyzed by qRT-PCR and Western blot. The correlations between Brachyury expression and clinicopathological factors of breast cancer were determined. Involvement of EMT stimulation and hypoxia-inducible factor-1α (HIF-1α) expression induction by Brachyury was also evaluated. Moreover, the effect of Brachyury on tumor growth and metastasis in vivo was examined in a breast tumor xenograft model. Brachyury expression was enhanced in primary breast cancer tissues and Brachyury expression was correlated with tumor stage and lymph node metastasis. Hypoxia enhanced Brachyury expression, the silencing of which blocked the modulation effect of hypoxia on E-cadherin and vimentin expression. Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling as well as accelerated cell proliferation and migration in vitro. Additionally, Brachyury accelerated breast tumor xenograft growth and increased lung metastasis in nude mice. In summary, our data confirmed that Brachyury might contribute to hypoxia-induced EMT of breast cancer and trigger HIF-1alpha expression via PTEN/Akt signaling. - Highlights: • Brachyury expression was correlated with tumor stage and lymph node metastasis. • Hypoxia enhanced Brachyury expression, which contributes to hypoxia-induced EMT. • Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling. • Brachyury accelerated tumor xenograft growth and increased lung metastasis.

  17. Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary Epithelial Cell Differentiation

    Science.gov (United States)

    2007-03-01

    Kobayashi, R. & Stillman, B. Characterization of the five replication factor C genes of Saccharomyces cerevisiae . Mol. Cell Biol. 15, 4661-4671... Saccharomyces cerevisiae . Mol. Cell. Biol. 15, 4661–4671. Davila, M., Frost, A.R., Grizzle, W.E., and Chakrabarti, R. (2003). LIM kinase 1 is essential for...for supplying nutrition as well as removing waste, consistent with the observation that ANG1 causes vessel enlargement without angiogenic sprouting

  18. Inhibition of Breast Cancer Progression by Blocking Heterocellular Contact Between Epithelial Cells and Fibroblasts

    Science.gov (United States)

    2013-04-01

    cells were cultured in flasks with low-glucose DMEM (1.0 mg mL!1, Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS) and 1...cultured in high glucose DMEM (4.5 mg mL!1, Gibco), supplemented with 10% calf serum (CS) and 1% penicillin/ streptomycin. Both cell lines were cultured in...were re-suspended in serum -free DMEM (SF-DMEM). FN or LN were mixed with the neutralized CN, 1.5% (v/v) calf serum , and SF-DMEM to get final ECM

  19. Effects of Environmental Estrogens on Apoptosis in Normal and Cancerous Breast Epithelial Cells

    Science.gov (United States)

    2001-03-01

    5325-5328, 1996. Gamen, S., Marzo, I., Anel , A., Pineiro, A., and Naval, J. CPP32 inhibition prevents Fas-induced ceramide generation and apoptosis...Herbst linked the occurrence of rare cervical and vaginal can- cers in a small group of young women who were treated by him at the Massachusetts General...circulation, across the placenta, into the fetal circulation and causes a change in the developing vaginal cell, resulting in cancer after the developing

  20. The histone H2A isoform Hist2h2ac is a novel regulator of proliferation and epithelial-mesenchymal transition in mammary epithelial and in breast cancer cells.

    Science.gov (United States)

    Monteiro, Fátima Liliana; Vitorino, Rui; Wang, Jun; Cardoso, Hugo; Laranjeira, Hugo; Simões, Joana; Caldas, Margarida; Henrique, Rui; Amado, Francisco; Williams, Cecilia; Jerónimo, Carmen; Helguero, Luisa A

    2017-06-28

    Proliferation and differentiation are controlled through chromatin remodelling. Therefore, there is an enormous biological significance and clinical value in understanding how specific signalling pathways are affected by histone replacement in the nucleosome. In this work, mass spectrometry was used to screen HC11 mammary epithelial cells for changes in histone levels throughout cell differentiation. The canonical histone isoform Histone H2A type 2-C (Hist2h2ac) was found only in undifferentiated/proliferating cells. Hist2h2ac mRNA was induced by EGF, specifically in the CD24+/CD29hi/DC44hi cell subpopulation. Hist2h2ac mRNA was increased by MEK1/2 or PI3-K activation in HC11 and EpH4 mammary epithelial cells, and in MC4-L2 and T47-D breast cancer cells. Hist2h2ac silencing inhibited EGF-induced Zeb-1 expression and E-cadherin down-regulation, and this effect was reverted by Hist2h2ac re-expression. Notably, silencing of Hist2h2ac increased EGFR, ERBB2, and ERK1/2 activation but did not allow EGF-induced proliferation. HIST2H2AC was expressed in all breast cancer molecular subtypes and found altered in 17% breast cancers, being 16.8% of the cases related to HIST2H2AC gene amplification and/or mRNA upregulation. In summary, this is the first study that identifies a canonical histone isoform -Hist2h2ac-downstream of the EGFR pathway, regulating oncogenic signalling and thereby contributing to deregulation of target genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Soy isoflavone genistein modulates cell cycle progression and induces apoptosis in HER-2/neu oncogene expressing human breast epithelial cells.

    Science.gov (United States)

    Katdare, Meena; Osborne, Michael; Telang, Nitin T

    2002-10-01

    In the multistep progressive pathogenesis of human breast cancer, comedo ductal carcinoma in situ (DCIS) represents a preinvasive precursor lesion for therapy resistant invasive cancer. Human tissue derived cell culture models exhibiting molecular similarities to clinical DCIS facilitate an important preclinical mechanistic approach for evaluation of preventive efficacy of natural and synthetic chemopreventive compounds. Natural phytochemicals present in fresh fruits, vegetables and grain products are likely to offer protection against cancer. The clinical efficacy of these natural phytochemicals, however, depends on extrapolation, and is therefore equivocal. The present study determined whether the natural soy isoflavone genistein (GEN) inhibited aberrant proliferation in 184-B5/HER cells (a model for human comedo DCIS) and identified possible mechanisms responsible for its efficacy. Human reduction mammoplasty derived HER-2/neu oncogene expressing preneoplastic 184-B5/HER cells represented the experimental system. Flow cytometry and cellular epifluorescence based assays were utilized to quantitate the alterations in cell cycle progression, cellular apoptosis, and in the status of cell cycle regulatory and apoptosis-associated gene product expression. The 184-B5/HER cells exhibited specific immunofluorescence to p185HER, p53, EGFR, but not to ERalpha, thus resembling comedo DCIS. Treatment of 184-B5/HER cells with GEN resulted in a dose-dependent decrease in the viable cell population, increase in the G0/G1:S + G2/M ratio and enhancement of sub G0/G1 (apoptotic population). Exposure to the maximum cytostatic 10 microM dose of GEN down-regulated HER-2/neu mediated signal transduction as evidenced by a 73.9% decrease (p=0.001) in p185HER specific, and a 89.8% decrease (p=0.001) in phosphotyrosine specific immunofluorescence. The increase in G0/G1:S + G2/M ratio in response to the treatment with 10 microM GEN was associated with a 85.5% decrease (p=0.001) in

  2. MHP-1 inhibits cancer metastasis and restores topotecan sensitivity via regulating epithelial-mesenchymal transition and TGF-β signaling in human breast cancer cells.

    Science.gov (United States)

    Lin, Sensen; Lyu, Xiaodan; Yu, Jun; Sun, Li; Du, Danyu; Lai, Yanqi; Li, Hongyang; Wang, Ying; Zhang, Luyong; Yin, Hongping; Yuan, Shengtao

    2016-09-15

    Cordyceps has long been used to treat cancer. However, its pharmacologically active components as well as the molecular mechanisms underlying its effects are still unclear. To investigate the effect of MHP-1, a newly isolated polysaccharide from Mortierella hepialid (the asexual structure of C. sinensis), on breast cancer metastasis. The effect of MHP-1 on breast cancer cell migration, epithelial-mesenchymal transition (EMT) and TGF-β signaling were investigated in vitro and in vivo. The effect of MHP-1 against topotecan-resistant MCF-7 cells that developed an EMT-like phenotype was also examined. The in vitro effect of MHP-1 on breast cancer cell proliferation and migration was evaluated by CCK8 and transwell assay. Morphological changes were observed and EMT markers were detected by western blot. The production of MMPs was measured by quantitative PCR and ELISA assay. To further investigate the mechanism that MHP-1 inhibited breast cancer EMT, western blot, ELISA, luciferase reporter gene assay, siRNA, quantitative PCR, immunohistochemistry, and xenograft tumor model were performed. MHP-1 inhibited breast cancer cell migration but did not cause any cytotoxicity. MHP-1 significantly surpressed breast cancer EMT, and slightly decreased MMP-9 secretion. TGF-β signaling was selectively inhibited after MHP-1 treatment, and other EMT-related pathways, like Wnt and Notch, were not affected. MHP-1 reduced the secretion of TGF-β1, but rarely affected other EMT-induced cytokines. Dual luciferase assay and Smad2/3 phosphorylation analysis indicated that MHP-1 suppressed TGF-β signaling. We further showed that MHP-1 restored sensitivity in topotecan (TPT)-resistant MCF-7 cells that developed an EMT-like phenotype. Similarly, the effect of TPT on resistant MCF-7 cells was also increased either by ALK5 (TGFβRI) siRNA or by a small molecular inhibitor of ALK5, SB-431542. MHP-1 inhibited breast cancer metastasis in the MDA-MB-231 xenograft model, and the immunohistochemical

  3. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells.

    Science.gov (United States)

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  4. In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis.

    Science.gov (United States)

    Bulfoni, Michela; Gerratana, Lorenzo; Del Ben, Fabio; Marzinotto, Stefania; Sorrentino, Marisa; Turetta, Matteo; Scoles, Giacinto; Toffoletto, Barbara; Isola, Miriam; Beltrami, Carlo Alberto; Di Loreto, Carla; Beltrami, Antonio Paolo; Puglisi, Fabio; Cesselli, Daniela

    2016-03-09

    Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data. This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively. By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly

  5. Anti-estrogen resistance in breast cancer is induced by the tumor microenvironment and can be overcome by inhibiting mitochondrial function in epithelial cancer cells.

    Science.gov (United States)

    Martinez-Outschoorn, Ubaldo E; Goldberg, Allison; Lin, Zhao; Ko, Ying-Hui; Flomenberg, Neal; Wang, Chenguang; Pavlides, Stephanos; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

    2011-11-15

    mechanism for conferring drug resistance. In summary, we have discovered that mitochondrial activity in epithelial cancer cells drives tamoxifen resistance in breast cancer and that mitochondrial "poisons" are able to re-sensitize these cancer cells to tamoxifen. In this context, TIGAR may be a key "druggable" target for preventing drug resistance in cancer cells, as it protects cancer cells against the onset of stress-induced mitochondrial dys-function and aerobic glycolysis.

  6. Membrane-initiated estradiol signaling of epithelial-mesenchymal transition-associated mechanisms through regulation of tight junctions in human breast cancer cells.

    Science.gov (United States)

    Jiménez-Salazar, Javier E; Posadas-Rodríguez, Pedro; Lazzarini-Lechuga, Roberto C; Luna-López, Armando; Zentella-Dehesa, Alejandro; Gómez-Quiroz, Luis E; Königsberg, Mina; Domínguez-Gómez, Guadalupe; Damián-Matsumura, Pablo

    2014-06-01

    Tumor cells utilize inappropriate epithelial-mesenchymal transition (EMT) mechanisms during the invasive process. It is becoming increasingly clear that estradiol (E2) induces breast cancer cell progression and enhances EMT; however, the mechanisms associated with this are unclear. We investigated the role of E2 on the expression and intracellular localization of the tight junction (TJ)-associated proteins, zonula occluden 1 (ZO-1), ZO-1-associated nucleic acid binding (ZONAB), and occludin, on the activation of c-Src and human epidermal growth factor receptor 2 (HER2) expression and cellular migration in the estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. We demonstrated that 1 nM E2 elicits c-Src activation after 15 min. The p-Src/ZO-1 complex led to ZO-1 and ZONAB disruption at the TJ and increased expression of HER2 mRNAs. These changes correlate with decreased expression of the epithelial markers occludin and CRB3 and increased synthesis of N-cadherin. This led to increased MCF-7 cell migration induced by E2, even in the presence of a cell proliferation inhibitor. Incubation with ICI 182,780 (Fulvestrant), an ER antagonist, precluded the effects of E2 on c-Src phosphorylation, p-Src/ZO-1 complex formation, ZO-1/ZONAB nuclear translocation, and migration of MCF-7 cells. Our findings suggest that E2 promotes TJ disruption during tumor progression and increases cell motility. We propose a novel pathway where estrogens promote EMT-associated mechanisms that possibly lead to metastasis.

  7. Expression, regulation and function of epithelial-stromal interaction 1 (breast), EPSTI1

    DEFF Research Database (Denmark)

    Villadsen, René; Lind Nielsen, Helga; Rank, Fritz

    2008-01-01

    The transcript of epithelial-stromal interaction 1 (breast), EPSTI1, was originally found to be upregulated in invasive breast carcinomas as compared to normal breast tissue (Genomics 79:703, 2002). In the present study we investigated the expression, regulation and possible function of the protein...... in epithelial cells in close contact with stromal cells. Furthermore, preliminary data indicate that the expression was highest in estrogen receptor negative tumors. By comparison, in normal breast EPSTI1 was expressed primarily in the acini. To address a possible function of the protein we used a human breast...... performed immunocytochemistry on a human breast epithelial cell line exposed to interferon-a. EPSTI1 as well as STAT1 were induced. Specific downregulation of EPSTI1 message with siRNAs also led to lower expression of á2â1-integrin. In conclusion, we have confirmed the existence of a protein product...

  8. A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer.

    Science.gov (United States)

    von Eyss, Björn; Jaenicke, Laura A; Kortlever, Roderik M; Royla, Nadine; Wiese, Katrin E; Letschert, Sebastian; McDuffus, Leigh-Anne; Sauer, Markus; Rosenwald, Andreas; Evan, Gerard I; Kempa, Stefan; Eilers, Martin

    2015-12-14

    In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues

    Directory of Open Access Journals (Sweden)

    Buršíková Eva

    2008-04-01

    Full Text Available Abstract Background Breast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences. Methods We prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE gels in pH range 4–7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package and data-mining analysis (GUHA. For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS was employed. Results Three protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased. Conclusion We have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.

  10. microRNA alterations in ALDH positive mammary epithelial cells: a crucial contributing factor towards breast cancer risk reduction in case of early pregnancy.

    Science.gov (United States)

    Nandy, Sushmita Bose; Subramani, Ramadevi; Rajamanickam, Venkatesh; Lopez-Valdez, Rebecca; Arumugam, Arunkumar; Boopalan, Thiyagarajan; Lakshmanaswamy, Rajkumar

    2014-08-31

    microRNAs have recently succeeded in grabbing the center stage in cancer research for their potential to regulate vital cellular process like cell cycle, stem cell renewal and epithelial mesenchymal transition. Breast cancer is the second most leading cause of cancer related mortality in women. The main reason for mortality is chemoresistance and metastasis for which remnant stem cells are believed to be the cause. One of the natural ways to reduce the risk of breast cancer in women is early pregnancy. Unraveling the mechanism behind it would add to our knowledge and help in evolving newer paradigms for breast cancer prevention.The current study deals with investigating transcriptomic differences in putative stem cells in mammary epithelial cell population (MECs) in terms of genes and microRNAs. In silico tools were used to identify potential mechanisms. ALDH positive MECs represent a putative stem cell population in the mammary gland. MECs were extracted from the mammary gland of virgin and parous (one time pregnant) rats. ALDH positive MECs were sorted and used for transcriptional and translational analysis for genes and microRNAs. In silico analysis for target prediction and networking was performed through online portals of Target Scan and Metacore. A total of 35 and 49 genes and microRNAs respectively were found to be differentially expressed within the two groups. Among the important genes were Lifr, Acvr1c, and Pparγ which were found to be targeted by microRNAs in our dataset like miR-143, miR-30, miR-140, miR-27b, miR-125a, miR-128ab, miR-342, miR-26ab, miR-181, miR-150, miR-23ab and miR-425. In silico data mining and networking also demonstrates that genes and microRNA interaction can have profound effects on stem cell renewal, cell cycle dynamics and EMT processes of the MEC population. Our data clearly shows that certain microRNAs play crucial role in the regulation of ALDH positive MECs and favor an anti-carcinogenic environment in the post

  11. Effects of sodium saccharin and linoleic acid on mRNA levels of Her2/neu and p53 in a human breast epithelial cell line.

    Science.gov (United States)

    Ogretmen, B; Ratajczak, H; Gendel, S M; Stark, B C

    1996-04-19

    The effects of two food-related chemicals (sodium saccharin and linoleic acid) on the levels of Her2/neu and p53 mRNA in a non-cancerous human breast epithelial cell line (HBL-100) were tested in comparison with the effects of the known tumor promoter phorbol 12-myristate 13-acetate (TPA). Treatments were made both with and without prior treatment with two well-known tumor initiators, N-nitroso-N-methylurea (NMU) or 7,12-dimethylbenz[a]anthracene (DMBA). The effects in general were small, the greatest being increases of 46-67% in Her2/neu mRNA levels in response to treatments with TPA or sodium saccharin following NMU treatments. These results demonstrate that sodium saccharin following NMU treatments might be involved in transcriptional regulation of Her2/neu in HBL-100 cells and suggest that its effects may not be limited to urinary bladder.

  12. GATA3 targets semaphorin 3B in mammary epithelial cells to suppress breast cancer progression and metastasis.

    Science.gov (United States)

    Shahi, P; Wang, C-Y; Chou, J; Hagerling, C; Gonzalez Velozo, H; Ruderisch, A; Yu, Y; Lai, M-D; Werb, Z

    2017-10-05

    Semaphorin 3B (SEMA3B) is a secreted axonal guidance molecule that is expressed during development and throughout adulthood. Recently, SEMA3B has emerged as a tumor suppressor in non-neuronal cells. Here, we show that SEMA3B is a direct target of GATA3 transcriptional activity. GATA3 is a key transcription factor that regulates genes involved in mammary luminal cell differentiation and tumor suppression. We show that GATA3 relies on SEMA3B for suppression of tumor growth. Loss of SEMA3B renders GATA3 inactive and promotes aggressive breast cancer development. Overexpression of SEMA3B in cells lacking GATA3 induces a GATA3-like phenotype and higher levels of SEMA3B are associated with better cancer patient prognosis. Moreover, SEMA3B interferes with activation of LIM kinases (LIMK1 and LIMK2) to abrogate breast cancer progression. Our data provide new insights into the role of SEMA3B in mammary gland and provides a new branch of GATA3 signaling that is pivotal for inhibition of breast cancer progression and metastasis.

  13. EGFR signaling pathways are wired differently in normal 184A1L5 human mammary epithelial and MDA-MB-231 breast cancer cells.

    Science.gov (United States)

    Speth, Zachary; Islam, Tanzila; Banerjee, Kasturi; Resat, Haluk

    2017-03-29

    Because of differences in the downstream signaling patterns of its pathways, the role of the human epidermal growth factor family of receptors (HER) in promoting cell growth and survival is cell line and context dependent. Using two model cell lines, we have studied how the regulatory interaction network among the key proteins of HER signaling pathways may be rewired upon normal to cancerous transformation. We in particular investigated how the transcription factor STAT3 and several key kinases' involvement in cancer-related signaling processes differ between normal 184A1L5 human mammary epithelial (HME) and MDA-MB-231 breast cancer epithelial cells. Comparison of the responses in these cells showed that normal-to-cancerous cellular transformation causes a major re-wiring of the growth factor initiated signaling. In particular, we found that: i) regulatory interactions between Erk, p38, JNK and STAT3 are triangulated and tightly coupled in 184A1L5 HME cells, and ii) STAT3 is only weakly associated with the Erk-p38-JNK pathway in MDA-MB-231 cells. Utilizing the concept of pathway substitution, we predicted how the observed differences in the regulatory interactions may affect the proliferation/survival and motility responses of the 184A1L5 and MDA-MB-231 cells when exposed to various inhibitors. We then validated our predictions experimentally to complete the experiment-computation-experiment iteration loop. Validated differences in the regulatory interactions of the 184A1L5 and MDA-MB-231 cells indicated that instead of inhibiting STAT3, which has severe toxic side effects, simultaneous inhibition of JNK together with Erk or p38 could be a more effective strategy to impose cell death selectively to MDA-MB-231 cancer cells while considerably lowering the side effects to normal epithelial cells. Presented analysis establishes a framework with examples that would enable cell signaling researchers to identify the signaling network structures which can be used to

  14. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  15. Beta-elemene blocks epithelial-mesenchymal transition in human breast cancer cell line MCF-7 through Smad3-mediated down-regulation of nuclear transcription factors.

    Directory of Open Access Journals (Sweden)

    Xian Zhang

    Full Text Available Epithelial-mesenchymal transition (EMT is the first step required for breast cancer to initiate metastasis. However, the potential of drugs to block and reverse the EMT process are not well explored. In the present study, we investigated the inhibitory effect of beta-elemene (ELE, an active component of a natural plant-derived anti-neoplastic agent in an established EMT model mediated by transforming growth factor-beta1 (TGF-β1. We found that ELE (40 µg/ml blocked the TGF-β1-induced phenotypic transition in the human breast cancer cell line MCF-7. ELE was able to inhibit TGF-β1-mediated upregulation of mRNA and protein expression of nuclear transcription factors (SNAI1, SNAI2, TWIST and SIP1, potentially through decreasing the expression and phosphorylation of Smad3, a central protein mediating the TGF-β1 signalling pathway. These findings suggest a potential therapeutic benefit of ELE in treating basal-like breast cancer.

  16. MiR-145 regulates epithelial to mesenchymal transition of breast cancer cells by targeting Oct4.

    Directory of Open Access Journals (Sweden)

    Jiajia Hu

    Full Text Available MiR-145 could regulate tumor growth, apoptosis, migration, and invasion. In our present study, we investigated its role in epithelial-mesenchymal transition (EMT. Expression of miR-145 was decreased in breast tumor tissues at T3&4 stages in comparison with those at T1&2. Over-expression of miR-145 mimics enhanced protein levels of E-cadherin and dampened those of α-SMA and Fibronectin, indicative of its inhibitory role in EMT occurrence. Mechanistic studies showed that miR-145 mimics inhibited Oct4 expression and miR-145 inhibitor enhanced it. Over-expression of Oct4 reversed miR-145-regulated expression of EMT markers, suggesting that Oct4 mediated the inhibitory effects of miR-145. MiR-145 could inhibite the expression of Snail, ZEB1, and ZEB2, while over-expression of Oct4 rescued the effects. Furthermore, Oct-4 induced over-expression of transcription factor Snail, ZEB1 and ZEB2 was mediated by β-catenin. Expression of Slug and Twist were not altered by miR-145/Oct4. Taken together, our results have revealed a novel role of miR-145 on EMT. It inhibits EMT by blocking the expression of Oct4, and downstream transcriptional factors, Snail, ZEB1 and ZEB2.

  17. Alterations in Galectin-3 Expression and Distribution Correlate with Breast Cancer Progression : Functional Analysis of Galectin-3 in Breast Epithelial-Endothelial Interactions

    OpenAIRE

    Shekhar, Malathy P.V.; Nangia-Makker, Pratima; Tait, Larry; Miller, Fred; Raz, Avraham

    2004-01-01

    To define the role of galectin-3 in breast cancer progression, we have used a novel three-dimensional co-culture system that recapitulates in vivo reciprocal functional breast epithelial-endothelial cell-cell and cell-matrix interactions, and examined the expression of galectin-3 mRNA and protein in human breast tumors and xenografts. Galectin-3 is required for the stabilization of epithelial-endothelial interaction networks because immunoneutralization with galectin-3 antibodies abolishes th...

  18. Downregulation of transcription factor Oct4 induces an epithelial-to-mesenchymal transition via enhancement of Ca{sup 2+} influx in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Jiajia; Qin, Kunhua; Zhang, Yan [Medical School of Nankai University, 94 Weijin Road, Tianjin 300071 (China); Gong, Junbo [Tianjin Key Laboratory of Modern Drug Delivery and High Efficiency in Tianjin University, 92 Weijin Road, Tianjin 300072 (China); Li, Na; Lv, Dan; Xiang, Rong [Medical School of Nankai University, 94 Weijin Road, Tianjin 300071 (China); Tan, Xiaoyue, E-mail: xiaoyuetan@nankai.edu.cn [Medical School of Nankai University, 94 Weijin Road, Tianjin 300071 (China)

    2011-08-12

    Highlights: {yields} We examine the role of Oct4 in metastasis in cultured MCF-7 cells. {yields} The down regulation of Oct4 induces EMT and increases the capability of migration and invasion in MCF-7 cells. {yields} TGF-{beta}1 inhibits Oct4 expression in both time- and dose-dependent manners. {yields} The EMT induced by TGF-{beta}1 or down regulation of Oct4 could be abrogated by inhibitor of SOCE. {yields} The down regulation of STIM1 (one of the major components of the CRAC channel) alleviates the EMT induce by Oct4 silencing down. -- Abstract: The stem cell-related transcription factor Oct4 regulates tumor proliferation and apoptosis, but its role in tumor migration and invasion is still undefined. Here, we compared Oct4 expression in MCF-7 and MDA-MB-231 cells, two breast cancer cell lines with similar epithelial origins, but distinct invasive and metastatic characteristics. We found MCF-7 cells to express very high levels of Oct4, while no obvious expression was detected in MDA-MB-231 cells. We then downregulated Oct4 expression using small interfering RNA (siRNA) to explore its effects on migration and invasion. Transwell assays showed that silencing Oct4 in MCF-7 cells improved their migration and invasion capabilities. Reverse-transcriptase PCR and western blots showed that E-cadherin expression decreased, and {alpha}-smooth muscle actin expression increased with Oct4 downregulation, which suggests that epithelial-to-mesenchymal transition (EMT) occurred. A potent EMT stimulus, TGF-{beta}1, significantly inhibited Oct4 expression in both dose- and time course-dependent manners. Silencing Oct4 also upregulated expression of two major components of store-operated Ca{sup 2+} entry channels (SOCs), STIM1 and Orai1, and enhanced SOC-directed Ca{sup 2+} influx. Silencing STIM1 blocked the Ca{sup 2+} influx and rescued the EMT initiated by Oct4 downregulation. In conclusion, silencing Oct4 promotes invasion and metastasis in breast cancer cells by inducing EMT

  19. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li JP

    2015-02-01

    , but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl and B-cell lymphoma 2 (Bcl-2, but increased the expression of Bcl-2-associated X protein (Bax and p53-upregulated modulator of apoptosis (PUMA, and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK and extracellular signal-regulated kinases 1 and 2 (Erk1/2 and inhibited the activation of protein kinase B (Akt/mammalian target of rapamycin (mTOR signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E

  20. Epithelial-mesenchymal transition markers and HER3 expression are predictors of elisidepsin treatment response in breast and pancreatic cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Cristina Teixidó

    Full Text Available Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734 is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT and the use of HER family receptors in predicting the in vitro drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1 and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.

  1. File list: DNS.Brs.05.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

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  16. Morphologic transformation of human breast epithelial cells MCF-10A: dependence on an oxidative microenvironment and estrogen/epidermal growth factor receptors

    Directory of Open Access Journals (Sweden)

    Yusuf Rita

    2010-09-01

    Full Text Available Abstract Background MCF-10A, immortalized but non-transformed human breast epithelial cells, are widely used in research examining carcinogenesis. The studies presented here were initiated with the observation that MCF-10A cells left in continuous culture for prolonged periods without re-feeding were prone to the development of transformed foci. We hypothesized that the depletion of labile culture components led to the onset of processes culminating in the observed cell transformation. The purpose of this study was to define the factors which promoted transformation of this cell line. Results Changes in levels of phenol red (PHR, hydrocortisone (HC, and epidermal growth factor (EGF with or without estrogen treatment indicated that both oxidative stress- and estrogen receptor alpha (ERα-mediated pathways contribute to cell transformation. Gene array and Western blotting analyses of cells maintained in our laboratory and of those from other sources documented detectable ERα and ERbeta (ERβ in this ERα-negative cataloged cell line. Results also indicate the possibility of a direct association of EGF receptor (EGFR and ERα in these cells as well as the formation and high induction of a novel ternary complex that includes ERβ (ERα/ERβ/EGFR in cells grown under conditions facilitating transformation. Conclusions Our studies resulted in the development of a growth protocol where the effects of chronic, physiologically relevant alterations in the microenvironment on cellular transformation were examined. From our results, we were able to propose a model of transformation within the MCF-10A cell line in which oxidative stress, ER and EGFR play essential roles. Overall, our work indicates that the immediate microenvironment of cells exerts powerful growth cues which ultimately determine their transformation potential.

  17. Role of Cell Senescence in Breast Cancer

    National Research Council Canada - National Science Library

    Krtolica, Ana

    2000-01-01

    .... Here, we report that both mouse and human immortal pre-malignant breast epithelial cell lines increase 2 to 5 times their proliferation in the presence of senescent, compared to presenescent, human fibroblasts...

  18. Role of Cell Senescence in Breast Cancer

    National Research Council Canada - National Science Library

    Krtolica, Ana

    1999-01-01

    .... Here, we report that both mouse and human immortal pre-malignant breast epithelial cell lines show increased proliferation in the presence of senescent, compared to presenescent, human fibroblasts...

  19. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  20. Exploring the spatial dimension of estrogen and progesterone signaling: detection of nuclear labeling in lobular epithelial cells in normal mammary glands adjacent to breast cancer.

    Science.gov (United States)

    Grote, Anne; Abbas, Mahmoud; Linder, Nina; Kreipe, Hans H; Lundin, Johan; Feuerhake, Friedrich

    2014-01-01

    Comprehensive spatial assessment of hormone receptor immunohistochemistry staining in digital whole slide images of breast cancer requires accurate detection of positive nuclei within biologically relevant regions of interest. Herein, we propose a combination of automated region labeling at low resolution and subsequent detailed tissue evaluation of subcellular structures in lobular structures adjacent to breast cancer, as a proof of concept for the approach to analyze estrogen and progesterone receptor expression in the spatial context of surrounding tissue. Routinely processed paraffin sections of hormone receptor-negative ductal invasive breast cancer were stained for estrogen and progesterone receptor by immunohistochemistry. Digital whole slides were analyzed using commercially available image analysis software for advanced object-based analysis, applying textural, relational, and geometrical features. Mammary gland lobules were targeted as regions of interest for analysis at subcellular level in relation to their distance from coherent tumor as neighboring relevant tissue compartment. Lobule detection quality was evaluated visually by a pathologist. After rule set optimization in an estrogen receptor-stained training set, independent test sets (progesterone and estrogen receptor) showed acceptable detection quality in 33% of cases. Presence of disrupted lobular structures, either by brisk inflammatory infiltrate, or diffuse tumor infiltration, was common in cases with lower detection accuracy. Hormone receptor detection tended towards higher percentage of positively stained nuclei in lobules distant from the tumor border as compared to areas adjacent to the tumor. After adaptations of image analysis, corresponding evaluations were also feasible in hormone receptor positive breast cancer, with some limitations of automated separation of mammary epithelial cells from hormone receptor-positive tumor cells. As a proof of concept for object-oriented detection of

  1. Epithelial cell polarity, stem cells and cancer

    DEFF Research Database (Denmark)

    Martin-Belmonte, Fernando; Perez-Moreno, Mirna

    2011-01-01

    , deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.......After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related...

  2. Remodeling of purinergic receptor-mediated Ca2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Felicity M Davis

    Full Text Available BACKGROUND: The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT, may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. METHODOLOGY/PRINCIPAL FINDINGS: We assessed changes in intracellular Ca(2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR(TETRA and observed significant changes in the potency of ATP (EC(50 0.175 µM (-EGF versus 1.731 µM (+EGF, P<0.05, and the nature of the ATP-induced Ca(2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05. We observed no change in the sensitivity to PAR2-mediated Ca(2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca(2+ homeostasis. To determine whether changes in ATP-mediated Ca(2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X(5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X(5 leads to a significant reduction (25%, P<0.05 in EGF-induced vimentin protein expression. CONCLUSIONS: The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of

  3. Insulin-Like Growth Factor-1 Inscribes a Gene Expression Profile for Angiogenic Factors and Cancer Progression in Breast Epithelial Cells

    Directory of Open Access Journals (Sweden)

    J.S. Oh

    2002-01-01

    Full Text Available Activation of the insulin-like growth factor-1 receptor (IGF-11R by IGF-1 is associated with the risk and progression of many types of cancer, although despite this it remains unclear how activated IGF-1 R contributes to cancer progression. In this study, gene expression changes elicited by IGF-1 were profiled in breast epithelial cells. We noted that many genes are functionally linked to cancer progression and angiogenesis. To validate some of the changes observed, the RNA and/or protein was confirmed for c-fos, cytochrome P4501Al, cytochrome P450 1131, interleukin-1 beta, fas ligand, vascular endothelial growth factor, and urokinase plasminogen activator. Nuclear proteins were also temporally monitored to address how gene expression changes were regulated. We found that IGF-1 stimulated the nuclear translocation of phosphorylated AKT, hypoxic-inducible factor-1 alpha, and phosphorylated cAMP-responsive element-binding protein, which correlated with temporal changes in gene expression. Next, the promoter regions of IGF-1-regulated genes were searched in silico. The promoters of genes that clustered together had similar regulatory regions. In summary, IGF-1 inscribes a gene expression profile relevant to cancer progression, and this study provides insight into the mechanism(s whereby some of these changes occur.

  4. Trefoil factor 3 isolated from human breast milk downregulates cytokines (IL8 and IL6 and promotes human beta defensin (hBD2 and hBD4 expression in intestinal epithelial cells HT-29

    Directory of Open Access Journals (Sweden)

    Girolamo Jose Barrera

    2012-11-01

    Full Text Available Trefoil factors (TFF are secretory products of mucin producing cells. They play a key role in the maintenance of the surface integrity of oral mucosa and enhance healing of the gastrointestinal mucosa by a process called restitution. TFF comprises the gastric peptides (TFF1, spasmolytic peptide (TFF2, and the intestinal trefoil factor (TFF3. They have an important and necessary role in epithelial restitution within the gastrointestinal tract. Significant amounts of TFF are present in human milk. This study aimed to determine a possible correlation between TFF3 isolated from human breast milk and levels of cytokines (IL8 and IL6 and defensins (hBD2 and hBD4 in intestinal epithelial cells HT-29 treated with trefoil. Samples of human milk were collected within 2-4 weeks postpartum from healthy human mothers (18-30-years-old by manual breast massage, and TFF3 was purified by ammonium sulfate precipitation, isoelectric precipitation, DEAE-chromatography, and gel filtration. In this work we measured the concentrations and mRNA levels of cytokines and defensins by immunoassay (ELISA and semiquantitative RT-PCR technique, respectively. Also we measured the peroxidase activity. We present the first evidence of human milk TFF3 purification. Here we show that the presence of TFF3 isolated from milk strongly correlates with downregulation of IL8 and IL6 in human intestinal epithelial cells. On the other hand, TFF3 activated the epithelial cells in culture to produce beta defensins 2 (hBD2 and beta defensins 4 (hBD4. These findings suggest that TFF can activate intestinal epithelial cells and could actively participate in the immune system of breastfed babies by inducing the production of peptides related to innate defence, such as defensins.

  5. An in vitro model that recapitulates the epithelial to mesenchymal transition (EMT in human breast cancer.

    Directory of Open Access Journals (Sweden)

    Elad Katz

    Full Text Available The epithelial to mesenchymal transition (EMT is a developmental program in which epithelial cells down-regulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In human breast cancer, invasion into surrounding tissue is the first step in metastatic progression. Here, we devised an in vitro model using selected cell lines, which recapitulates many features of EMT as observed in human breast cancer. By comparing the gene expression profiles of claudin-low breast cancers with the experimental model, we identified a 9-gene signature characteristic of EMT. This signature was found to distinguish a series of breast cancer cell lines that have demonstrable, classical EMT hallmarks, including loss of E-cadherin protein and acquisition of N-cadherin and vimentin expression. We subsequently developed a three-dimensional model to recapitulate the process of EMT with these cell lines. The cells maintain epithelial morphology when encapsulated in a reconstituted basement membrane, but undergo spontaneous EMT and invade into surrounding collagen in the absence of exogenous cues. Collectively, this model of EMT in vitro reveals the behaviour of breast cancer cells beyond the basement membrane breach and recapitulates the in vivo context for further investigation into EMT and drugs that may interfere with it.

  6. Regulation of the O-glycan-type Sialyl-Lewis X (sLex) Bio-synthesis Pathway during Cell Transformation Programs: Epithelial-Mesenchymal Transition (EMT) and Molecular Subtypes in Breast Carcinoma and Human T Cell Activation

    KAUST Repository

    AbuElela, Ayman

    2017-12-01

    During tumor progression and development of distant metastases, a subset of cancer cells undergoes transformation programs, such as epithelial-mesenchymal transition (EMT), to acquire enhanced migratory attributes to commence the metastatic cascade with the intension of achieving an active cell adhesion molecule-mediated organ-specific homing. Similarly, naive T cells reform the assemblage of their surface adhesion molecules during differentiation to activated T cells in order to successfully home to sites of inflammation and other extra-lymphoid organs for surveillance purposes. Sialyl-Lewis X (sLex) is well-known for mediating the homing of epithelial circulating tumor cellss (CTCs) and activated T cells to target sites through the interaction with endothelial selectins. Since glycan structures are not directly encoded by the genome, their expression is dependent on the glycosyltransferase (GT) expression and activity. Yet, the modulation of GTs during breast cancer transformation and in different molecular subtypes is still unknown. In addition, although the regulation of GTs during T cell activation is well-understood, the regulation at the epigenetic level is lacking. O-glycan-type sLex expression and E-selectin binding under static and flow conditions varies among molecular subtypes of breast cancer and upon the induction of EMT which is linked to the expression patterns of GTs. GTs displayed a significant prognostic value of in the association with the patients\\' survival profiles and in the ability to predict the breast cancer molecular subtypes from the expression data of a random patient sample. Also, GTs were able to differentiate between tumor and their normal counterparts as well as cancer types and glioblastoma subtypes. On the other hand, we studied the regulation of GTs in human CD4+ memory T cells compared to the naive cells at the epigenetic level. Memory T cell subsets demonstrated differential chromatin accessibility and histone marks within

  7. Establishment of twist-1 and TGFBR2 as direct targets of microRNA-20a in mesenchymal to epithelial transition of breast cancer cell-line MDA-MB-231.

    Science.gov (United States)

    De, Soumasree; Das, Sayantani; Mukherjee, Srimoyee; Das, Sainy; Sengupta Bandyopadhyay, Sumita

    2017-12-01

    Messenchymal to epithelial transition (MET) is a significant physiological phenomenon involved in embryogenesis and cancer. This study aims at investigating the mechanism of microRNA-20a (miR-20a) mediated regulation of mesenchymal to epithelial transition and identification of its direct target genes in breast cancer cell-line, MDA-MB-231. Reduced migratory and invasive property, altered cellular morphology along with reduced capability for attachment to basement membrane was acquired by over-expression of miR-20a in invasive MDA-MB-231 cell-line initially expressing low level of this micro-RNA, indicating direct correlation between abundance of miR-20a and metastatic property. The switch from mesenchymal to epithelial cells mediated by miR-20a involved post-transcriptional down-regulation of twist1, which in turn controls downstream epithelial markers like E-cadherin, claudin and mesenchymal markers like N-cadherin, fibronectin, the crucial players of mesenchymal to epithelial transition (MET). Furthermore, another key component, TGF-β and one of its receptors (TGFBR2) were found to be down-regulated by miR-20a. Additionally, reporter assay established that post-transcriptional down-regulation of TGFBR2 occurred through direct binding of miR-20a to its 3'UTR, thus abrogating the TGF-β signaling pathway resulting in inhibition of MET. Delineating the underlying molecular mechanism of miR-20a-mediated MET and defining the target genes will help us to introduce a miRNA-mediated effective therapeutic strategy against breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Palbociclib inhibits epithelial-mesenchymal transition and metastasis in breast cancer via c-Jun/COX-2 signaling pathway

    OpenAIRE

    Qin, Ge; Fei XU; Qin, Tao; Zheng, Qiufan; Shi, Dingbo; Xia, Wen; Tian, Yun; Tang, Yanlai; Wang, Jingshu; Xiao, Xiangshen; Deng, Wuguo; Wang, Shusen

    2015-01-01

    Palbociclib, a highly selective CDK4/6 inhibitor, has been shown to be a novel anti-tumor agent that suppresses breast cancer cell proliferation. However, its anti-metastasis activity remains controversial. In the present study, we evaluated whether palbociclib prevented breast cancer cell metastasis and revealed its regulatory mechanism. We found that palbociclib inhibited migration and invasion in the breast cancer cells MDA-MB-231 and T47D. The epithelial-mesenchymal transition (EMT) marke...

  9. File list: NoD.Brs.20.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: InP.Brs.50.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Brs.50.AllAg.Mammary_epithelial_cells mm9 Input control Breast Mammary epitheli...al cells SRX424873,SRX403487,SRX396750,SRX031066,SRX031214 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Brs.50.AllAg.Mammary_epithelial_cells.bed ...

  11. File list: InP.Brs.20.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Brs.20.AllAg.Mammary_epithelial_cells mm9 Input control Breast Mammary epitheli...al cells SRX424873,SRX403487,SRX396750,SRX031066,SRX031214 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Brs.20.AllAg.Mammary_epithelial_cells.bed ...

  12. File list: InP.Brs.10.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: InP.Brs.05.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. Leptin-induced Epithelial-Mesenchymal Transition in Breast Cancer Cells Requires β-Catenin Activation via Akt/GSK3- and MTA1/Wnt1 Protein-dependent Pathways*

    Science.gov (United States)

    Yan, Dan; Avtanski, Dimiter; Saxena, Neeraj K.; Sharma, Dipali

    2012-01-01

    Perturbations in the adipocytokine profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1), and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with the small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion, and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of the GSK3β-LKB1-Axin complex that leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of the Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation, and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors shows increased expression of Wnt1, pGSK3β, and vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized cross-talk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells. PMID

  15. Eosinophils promote epithelial to mesenchymal transition of bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Atsushi Yasukawa

    Full Text Available Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

  16. A Novel Technique to Follow Consequences of Exogenous Factors, Including Therapeutic Drugs, on Living Human Breast Epithelial Cells

    Science.gov (United States)

    1999-07-01

    and lipid vectors, are being tested. Concurrent with the development of procedures for live - cell imaging , we are examining the distribution of proteins...dimensional matrix. These studies have not yet begun. There are a number of procedures that must be developed and perfected in the live - cell imaging , as...components of the Wnt signaling pathway are too preliminary and require additional research prior to publication. (9) CONCLUSIONS Live cell imaging of

  17. Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and β-catenin signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongzhong; Huang, Jing; Yang, Bing; Xiang, Tingxiu; Yin, Xuedong; Peng, Weiyan; Cheng, Wei [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Wan, Jingyuan; Luo, Fuling [Department of Pharmacology, Chongqing Medical University, Chongqing (China); Li, Hongyuan [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Ren, Guosheng, E-mail: rgs726@163.com [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China)

    2013-10-01

    Although mangiferin which is a naturally occurring glucosylxanthone has exhibited promising anticancer activities, the detailed molecular mechanism of mangiferin on cancers still remains enigmatic. In this study, the anticancer activity of mangiferin was evaluated in breast cancer cell line-based in vitro and in vivo models. We showed that mangiferin treatment resulted in decreased cell viability and suppression of metastatic potential in breast cancer cells. Further mechanistic investigation revealed that mangiferin induced decreased matrix metalloproteinase (MMP)-7 and -9, and reversal of epithelial–mesenchymal transition (EMT). Moreover, it was demonstrated that mangiferin significantly inhibited the activation of β-catenin pathway. Subsequent experiments showed that inhibiting β-catenin pathway might play a central role in mangiferin-induced anticancer activity through modulation of MMP-7 and -9, and EMT. Consistent with these findings in vitro, the antitumor potential was also verified in mangiferin-treated MDA-MB-231 xenograft mice where significantly decreased tumor volume, weight and proliferation, and increased apoptosis were obtained, with lower expression of MMP-7 and -9, vimentin and active β-catenin, and higher expression of E-cadherin. Taken together, our study suggests that mangiferin might be used as an effective chemopreventive agent against breast cancer. - Highlights: • Mangiferin inhibits growth and metastatic potential in breast cancer cells. • Mangiferin down-regulates MMP-7 and -9 in breast cancer cells. • Mangiferin induces the reversal of EMT in metastatic breast cancer cells. • Mangiferin inhibits the activation of β-catenin pathway in breast cancer cells. • Inhibiting β-catenin is responsible for the antitumor activity of mangiferin.

  18. Isolation and culture of biliary epithelial cells.

    OpenAIRE

    Joplin, R

    1994-01-01

    At one time it was thought that biliary epithelial cells simply formed the lining to the tubular conduits which constitute the biliary tract. Development of in vitro systems for culturing biliary epithelial cells has enabled functional studies which increasingly show that this is far from true, and that biliary epithelial cells do have important functional roles. Disruption of these functions may be involved in the generation of pathology. Most functional studies to date have utilised cells i...

  19. File list: ALL.Brs.50.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Brs.50.AllAg.Mammary_epithelial_cells mm9 All antigens Breast Mammary epithelia...SRX396750,SRX031066,SRX031214 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Brs.50.AllAg.Mammary_epithelial_cells.bed ...

  20. File list: ALL.Brs.05.AllAg.Mammary_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Brs.05.AllAg.Mammary_epithelial_cells mm9 All antigens Breast Mammary epithelia...SRX031066,SRX031214,SRX396750 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Brs.05.AllAg.Mammary_epithelial_cells.bed ...

  1. Obesity reversibly depletes the basal cell population and enhances mammary epithelial cell estrogen receptor alpha expression and progenitor activity.

    Science.gov (United States)

    Chamberlin, Tamara; D'Amato, Joseph V; Arendt, Lisa M

    2017-11-29

    Obesity is correlated with an increased risk for developing postmenopausal breast cancer. Since obesity rates continue to rise worldwide, it is important to understand how the obese microenvironment influences normal mammary tissue to increase breast cancer risk. We hypothesized that obesity increases the proportion of luminal progenitor cells, which are thought to be the cells of origin for the most common types of breast cancer, potentially leading to an increased risk for breast cancer. To study the obese microenvironment within the mammary gland, we used a high-fat diet mouse model of obesity and human breast tissue from reduction mammoplasty surgery. We identified changes in breast epithelial cell populations using flow cytometry for cell surface markers, in vitro functional assays and expression of markers on breast tissue sections. In both obese female mice and women, mammary epithelial cell populations demonstrated significant decreases in basal/myoepithelial cells, using either flow cytometry or cell-type-specific markers (SMA and p63). Estrogen receptor alpha (ERα) expression was significantly increased in luminal cells in obese mammary tissue, compared with control mice or breast tissue from lean women. Functional assays demonstrated significantly enhanced mammary epithelial progenitor activity in obese mammary epithelial cells and elevated numbers of ERα-positive epithelial cells that were co-labeled with markers of proliferation. Weight loss in a group of obese mice reversed increases in progenitor activity and ERα expression observed in obese mammary tissue. Obesity enhances ERα-positive epithelial cells, reduces the number of basal/myoepithelial cells, and increases stem/progenitor activity within normal mammary tissue in both women and female mice. These changes in epithelial cell populations induced by obesity are reversible with weight loss. Our findings support further studies to examine how obesity-induced changes in stem/progenitor cells

  2. A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells

    Directory of Open Access Journals (Sweden)

    Williams Briana

    2003-10-01

    Full Text Available Abstract Background In Arabidopsis thaliana, the gene Tousled encodes a protein kinase of unknown function, but mutations in the gene lead to flowering and leaf morphology defects. We have recently cloned a mammalian Tousled-Like Kinase (TLK1B and found that it phosphorylates specifically histone H3, in vitro and in vivo. We now report the effects that overexpression of a kinase-dead mutant of TLK1B mediates in a normal diploid cell line. Results Expression of a kinase-dead mutant resulted in reduction of phosphorylated histone H3, which could have consequences in mitotic segregation of chromosomes. When analyzed by FACS and microscopy, these cells displayed high chromosome number instability and aneuploidy. This phenomenon was accompanied by less condensed chromosomes at mitosis; failure of a number of chromosomes to align properly on the metaphase plate; failure of some chromosomes to attach to microtubules; and the occasional presentation of two bipolar spindles. We also used a different method (siRNA to reduce the level of endogenous TLK1, but in this case, the main result was a strong block of cell cycle progression suggesting that TLK1 may also play a role in progression from G1. This block in S phase progression could also offer a different explanation of some of the later mitotic defects. Conclusions TLK1 has a function important for proper chromosome segregation and maintenance of diploid cells at mitosis in mammalian cells that could be mediated by reduced phosphorylation of histone H3 and condensation of chromosomes, although other explanations to the phenotype are possible.

  3. Induction of the mesenchymal to epithelial transition by demethylation- activated microRNA-200c is involved in the anti-migration/invasion effects of arsenic trioxide on human breast cancer cells.

    Science.gov (United States)

    Si, Lu; Jiang, Fei; Li, Yuan; Ye, Xianqing; Mu, Juan; Wang, Xingxing; Ning, Shilong; Hu, Chunyan; Li, Zhong

    2015-09-01

    Breast cancer is a major health problem worldwide. Current standard practices for treatment of breast cancer are less than satisfactory because of high rates of metastasis. Arsenic trioxide (As(2)O(3)), which induces demethylation of DNA and causes apoptosis, has been used as an anti-tumor agent. Little is known, however, regarding its anti-metastatic effects. The microRNA-200c (miR-200c), which is frequently lowly expressed in triple negative breast cancers (TNBCs), inhibits metastasis by inducing the mesenchymal to epithelial transition (MET). Here, we report that As(2)O(3) attenuates the migratory and invasive capacities of breast cancer cells, MDA-MB-231 and BT-549. Notably, As(2)O(3) induces an MET in vitro and in vivo, as determined by the increased expression of the epithelial marker, E-cadherin and decreased expressions of mesenchymal markers, N-cadherin and vimentin. Moreover, As(2)O(3) up-regulates the expression of miR-200c through demethylation. Over-expression of miR-200c enhances the expression of E-cadherin and decreases the expressions of N-cadherin and vimentin. Further, in MDA-MB-231 cells exposed to As(2)O(3), knockdown of miR-200c blocks the As(2)O(3) -induced MET. Finally, in MDA-MB-231 and BT-549 cells exposed to As(2)O(3), knockdown of miR-200c decreases the As(2)O(3) -induced inhibition of the migratory and invasive capacities. By identifying a mechanism whereby As(2)O(3) regulates miR-200c and MET, the results establish the anti-migration/invasion potential of arsenic trioxide. © 2014 Wiley Periodicals, Inc.

  4. The basal epithelial marker P-cadherin associates with breast cancer cell populations harboring a glycolytic and acid-resistant phenotype.

    Science.gov (United States)

    Sousa, Bárbara; Ribeiro, Ana Sofia; Nobre, Ana Rita; Lopes, Nair; Martins, Diana; Pinheiro, Céline; Vieira, André Filipe; Albergaria, André; Gerhard, René; Schmitt, Fernando; Baltazar, Fátima; Paredes, Joana

    2014-10-01

    Cancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers.Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia. Immunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting. We demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly

  5. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

    National Research Council Canada - National Science Library

    Choi, Chul Hee; Lee, Jun Sik; Lee, Yoo Chul; Park, Tae In; Lee, Je Chul

    2008-01-01

    ... of A. baumannii outer membrane protein A (AbOmpA) in interactions with epithelial cells. A. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms...

  6. Differential diagnosis of benign epithelial proliferations and carcinomas of the breast using antibodies to cytokeratins.

    Science.gov (United States)

    Jarasch, E D; Nagle, R B; Kaufmann, M; Maurer, C; Böcker, W J

    1988-03-01

    The immunohistochemical reactivity on frozen sections of diverse benign and malignant epithelial proliferations of human breast tissue from 156 patients was examined using antibodies to different cytokeratins. Antibodies recognizing cytokeratins 18 and 19 reacted with luminal epithelial cells but not with myoepithelial cells of normal mammary gland, cystic disease, adenosis, papilloma, and fibroadenoma or with a subpopulation of proliferating cells in sclerosing adenosis and epitheliosis. These antibodies reacted with the tumor cells of all in situ and invasive carcinomas. KA1 antibody, which by one- and two-dimensional gel electrophoresis and immunoblotting was shown to bind preferentially to cytokeratin 14 in a complex with cytokeratin 5, reacted with the nonproliferating myoepithelium of normal gland, cystic disease, adenosis, papilloma, fibroadenoma, and in situ carcinoma; it also reacted with a subpopulation of proliferating cells in sclerosing adenosis and epitheliosis (papillomatosis) but was negative with the tumor cells of all preinvasive and most invasive carcinomas. In adenotic and epitheliotic proliferations, groups of cells were identified that reacted strongly with KA1 antibody in addition to antibodies to cytokeratins 18 and 19. The data are discussed with respect to epithelial cell heterogeneity in the breast. We show that by using such antibodies, benign epithelial proliferations are clearly distinguished from carcinomas.

  7. Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state.

    Science.gov (United States)

    Soady, Kelly; Smalley, Matthew J

    2012-09-10

    Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells.

  8. Epithelial periostin expression is correlated with poor survival in patients with invasive breast carcinoma.

    Directory of Open Access Journals (Sweden)

    Ga-Eon Kim

    Full Text Available Invasion and metastasis are direct causes of mortality in patients with breast cancer and require reciprocal interactions between cancer cells and the extracellular matrix (ECM. Periostin, a fasciclin-containing adhesive ECM glycoprotein, is frequently overexpressed in various types of human cancer, and its overexpression in cancer-associated stroma and/or cancer cells is usually associated with poor clinical outcomes. However, the expression of periostin in the successive steps of breast tumorigenesis and its association with outcome variables have not been well established in breast carcinoma. The present study aimed to assess the role of periostin alteration in breast tumorigenesis and evaluate the putative prognostic value of periostin as a function of its compartmentalization. Immunohistochemical staining with anti-periostin antibody was performed in a total of 300 patients (26 patients with normal breast tissues, 76 patients with ductal carcinoma in situ [DCIS], and 198 patients with invasive breast carcinoma [IBC] using tissue microarray. Periostin immunoreactivity was assessed in both epithelial tissue and the surrounding stromal compartment. The mRNA and protein expression of periostin were analyzed in 10 paired normal/invasive cancer frozen specimens by quantitative real time-polymerase chain reaction and western blot analysis, respectively. In cancer tissues, periostin mRNA and protein expression were increased compared with adjacent normal tissues. Both epithelial and stromal periostin staining scores significantly increased in a stepwise manner with disease progression from normal breast tissue to DCIS and IBC (P = 0.000 and 0.000, respectively. High epithelial and stromal periostin expression was observed in 109/189 (57.7% and 158/189 (83.6% cases of IBC, respectively. High epithelial periostin expression was more frequently observed in the distant metastatic relapse-positive group than in the distant metastatic relapse

  9. Epithelial Cells in Urine: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... page: https://medlineplus.gov/labtests/epithelialcellsinurine.html Epithelial Cells in Urine To use the sharing features on ... page, please enable JavaScript. What is an Epithelial Cells in Urine Test? Epithelial cells are a type ...

  10. Stem cells in the human breast

    DEFF Research Database (Denmark)

    Petersen, Ole William; Polyak, Kornelia

    2010-01-01

    The origins of the epithelial cells participating in the development, tissue homeostasis, and cancer of the human breast are poorly understood. However, emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner, these generate the two main...... mammary cell lineages, producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from...... nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area, whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides...

  11. Patterning bacterial communities on epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Dwidar

    Full Text Available Micropatterning of bacteria using aqueous two phase system (ATPS enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions.

  12. Identification of epithelial cells in bronchoalveolar lavage.

    Science.gov (United States)

    Finotto, S; Rado, V; Dal Vecchio, A; Milani, G; Fabbri, L M; Maestrelli, P

    1993-01-01

    1. Damage to the bronchial epithelium occurs after the inhalation of toxic substances and allergens, and through virus infections and it may lead to increased desquamation of epithelial cells in bronchoalveolar lavage (BAL). 2. In this study we compared two methods of staining the epithelial cells of BAL, the conventional cytochemical May Grunwald-Giemsa stain (MGG) and an immunocytochemical technique using a monoclonal antibody anti-human cytokeratin (CK) detected with APAAP immuno-alkaline phosphatase. BAL was obtained from 13 subjects and the epithelial cells were cytocentrifuged either immediately after collection (fraction A) or after washing (fraction B). 3. Higher percentages of epithelial cells were identified in fraction A with CK (20.0 +/- 5.1%) than in fraction A with MGG (11.2 +/- 2.3%), which recognized only ciliated epithelial cells. In fact a proportion of CK-positive cells (34%) in fraction A were not ciliated. Underestimation of epithelial cells by MGG compared to CK was more pronounced in fraction B (8.0 +/- 2.9% and 22.9 +/- 3.0%, respectively) as there was a relative loss of ciliated CK+ cells after washings. 4. These results suggest that immunocytochemical staining with an anti-cytokeratin monoclonal antibody is more sensitive than using the MGG stain in detecting epithelial cells in BAL.

  13. In situ localization of tumor cells associated with the epithelial-mesenchymal transition marker Snail and the prognostic impact of lymphocytes in the tumor microenvironment in invasive ductal breast cancer.

    Science.gov (United States)

    Alkatout, Ibrahim; Hübner, Friederike; Wenners, Antonia; Hedderich, Jürgen; Wiedermann, Meike; Sánchez, Cristina; Röcken, Christoph; Mathiak, Micaela; Maass, Nicolai; Klapper, Wolfram

    2017-04-01

    Tumor surgery is aimed at complete resection of the lesion while ensuring a sufficient tumor-specific safety distance. Nevertheless, in many cases the most peripheral part - the invasion front - remains in situ. Tumor cells at the tumor margin have been reported to lose their epithelial properties and acquire features of mesenchymal cells. The process of epithelial-to-mesenchymal transition (EMT) is believed to be of prime importance for tissue and vessel invasion. Furthermore, the detection of tumor-infiltrating lymphocytes in the microenvironment of breast cancer might serve as a reliable prognostic marker. We investigated tissue microarrays of 352 breast cancer patients with regard to the presence and distribution of the EMT factor Snail, and the presence of FoxP3, CD3 and CD8 in the immune microenvironment. The expression of the transcription factor Snail is strongly associated with longer disease-free and overall survival. The presence of CD3, CD8 or FoxP3 is associated with a better outcome, although statistically significant results were noted only for FoxP3. The prognostic significance of FoxP3 and Snail were also proven in multivariate analysis. Based on previous studies concerning the intratumoral heterogeneity of EMT, our results suggest that Snail and FoxP3 are possible prognostic markers for breast cancer. The diverse presence of lymphocytes in the tumor microenvironment (CD3 and CD8) was confirmed. Although the importance of these markers is known, their specific role in tumor invasion and metastasis as well as their hierarchical organization in these tumors remain unclear. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Surface differentiation antigens of human mammary epithelial cells carried on the human milk fat globule.

    Science.gov (United States)

    Ceriani, R L; Thompson, K; Peterson, J A; Abraham, S

    1977-02-01

    Rabbit antibodies against components of the human milk fat globule bind specifically to normal human breast epithelial cells and cell lines derived from breast carcinomas, as well as to the outer surface of the human milk fat globule. Variation in indirect immunofluorescence staining in both intensity per cell and percentage of cells stained is observed for the different brest cell lines. Cells derived from other epithelial and other ectodermal tissues, fetal fibroblasts, cells of the blood buffy coat, and even fibroblasts of the breast itself do not bind the antibodies. This suggests that these antibodies are detecting cell-type-specific antigens. These normal breast epithelial cell antigens are on the cell surface and their expression is stable in long-term cultured cell lines, even after much chromosomal variation in a given line. By affinity chromatography, three distinct antigenic components can be isolated from the milk fat globule, one of which contains carbohydrate. These differentiation antigens of the human breast epithelial cell are not only useful as specific cell-type markers, but also can provide a tool to study the role of the cell surface in normal and neoplastic mammary development.

  15. Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21(Waf1/Cip1) mediated inhibition of Hh-Gli signaling.

    Science.gov (United States)

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Siddharth, Sumit; Das, Dipon; Nayak, Anmada; Kundu, Chanakya Nath

    2015-09-01

    Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC50 value was noted in cells treated with the combination of resveratrol (3μM) and curcumin (3μM) in comparison to 30μM of resveratrol or curcumin alone. Resveratrol+curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21(Waf/Cip1) in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21(Waf/Cip1) knockout cells suggests this combination caused apoptosis through p21(Waf/Cip1). Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2(Waf/Cip1) mediated inhibition of Hedgehog-Gli cascade. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    Energy Technology Data Exchange (ETDEWEB)

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India); Choudhuri, Tathagata [Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023 (India); Department of Biotechnology, Visva Bharati University, Santiniketan, West Bengal (India); Kundu, Chanakya Nath, E-mail: cnkundu@gmail.com [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India)

    2014-03-15

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  17. Problems of diagnostics and treatment of the epithelial and non-epithelial breast tumors

    Directory of Open Access Journals (Sweden)

    Van Shu

    2017-01-01

    Full Text Available Study objective. To improve diagnostics quality and surgical treatment efficiency in patients with non-epithelial and fibroepithelial malignant breast tumors.Materials and methods. The non-randomized study included 87 patients, among them 16 patients with breast sarcomas and 71 patients with phyllodes tumors of the breast. Primary diagnosis was made based on mammography, ultrasound scan (including contrast-enhanced ultrasound, and sonoelastography data. Organ-preserving surgeries were performed in 48 (67.6 % patients with phyllodes tumors and 2 (13 % patients with sarcomas, mastectomy and breast amputations were performed in 23 (32.4 % patients with phyllodes tumors and 14 (87 % patients with sarcomas. Follow-up duration varied between 3 and 132 months.Results and discussion. During routine examination of patients with phyllodes tumors and sarcomas diagnostic errors were made in 62 and 20 % of cases, respectively. Sonoelastography and contrast-enhanced ultrasound allowed to make a correct diagnosis in the majority of patients (70 %. Long-term treatment results correlated with tumor type. Over all survival for benign phyllodes tumors was significantly higher (97.6 % than overall survival for its malignant type (57 % and breast sarcomas (60 %. Sparing tumor removal (tumorectomy was associated with high incidence of local recurrences for all types of non-epithelial and fibroepithelial tumors (between 31 % and 100 %. Segmental resection was also associated with high incidence of local recurrences, especially for the malignant type of phyllodes tumor (83 %.Conclusion. Primary diagnostics of breast diseases should involve sonoelastography and a contrast-enhanced ultrasound scan. Surgical treatment for malignant phyllodes tumors and sarcomas should include breast amputation or mastectomy.

  18. Cytoplasmic Localization of Proline, Glutamic Acid, Leucine-rich Protein 1 (PELP1) Induces Breast Epithelial Cell Migration through Up-regulation of Inhibitor of κB Kinase ϵ and Inflammatory Cross-talk with Macrophages.

    Science.gov (United States)

    Girard, Brian J; Knutson, Todd P; Kuker, Bethanie; McDowell, Laura; Schwertfeger, Kathryn L; Ostrander, Julie H

    2017-01-06

    Cytoplasmic localization of proline, glutamic acid, leucine-rich protein 1 (PELP1) is observed in ∼40% of women with invasive breast cancer. In mouse models, PELP1 overexpression in the mammary gland leads to premalignant lesions and eventually mammary tumors. In preliminary clinical studies, cytoplasmic localization of PELP1 was seen in 36% of women at high risk of developing breast cancer. Here, we investigated whether cytoplasmic PELP1 signaling promotes breast cancer initiation in models of immortalized human mammary epithelial cells (HMECs). Global gene expression analysis was performed on HMEC lines expressing vector control, PELP1-wt, or mutant PELP1 in which the nuclear localization sequence was altered, resulting in cytoplasmic localization of PELP1 (PELP1-cyto). Global gene expression analysis identified that PELP1-cyto expression in HMECs induced NF-κB signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of κB kinase ϵ (IKKϵ) and increased phosphorylation of the NF-κB subunit RelB. To determine whether secreted factors produced by PELP1-cyto HMECs promote macrophage activation, THP-1 macrophages were treated with HMEC-conditioned medium (CM). PELP1-cyto CM induced changes in THP-1 gene expression as compared with control cell CM. Double conditioned medium (DCM) from the activated THP-1 cells was then applied to HMECs to determine whether paracrine signaling from PELP1-cyto-activated macrophages could in turn promote migration of HMECs. PELP1-cyto DCM induced robust HMEC migration, which was reduced in DCM from PELP1-cyto HMECs expressing IKKϵ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKϵ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. MAGEC2, an epithelial-mesenchymal transition inducer, is associated with breast cancer metastasis.

    Science.gov (United States)

    Yang, Fan; Zhou, Xingchun; Miao, Xia; Zhang, Tao; Hang, Xiaojun; Tie, Ru; Liu, Nan; Tian, Fei; Wang, Fuli; Yuan, Jianlin

    2014-05-01

    MAGEC2 is a member of melanoma antigen (MAGE) family of cancer-testis antigens and associated with tumor relapse and metastasis. Here, we investigated the expression of MAGEC2 in patients with breast cancer and its clinical effects with underlying mechanisms. The expression levels of MAGEC2 were compared between 420 invasive ductal carcinoma (IDC) and 120 ductal carcinoma in situ of the breast. Correlations between MAGEC2 expression and clinico-pathologic factors or survival of patients with IDC were analyzed. In addition, MAGEC2 expression levels in tumor tissues dissected from the primary focus and matched tumor-invaded axillary lymph nodes were analyzed in 8 breast cancer patients. The functional effects of MAGEC2 overexpression were assessed in vitro using scratch assay and transwell chamber assay. MAGEC2 expression was increased in metastatic breast cancer in comparison to the non-metastatic. MAGEC2 expression was significantly associated with ER negative expression (P = 0.037), high tumor grade (P = 0.014) and stage (P = 0.002), high incidence of axillary lymph node metastasis (P = 0.013), and distant metastasis (P = 0.004). Patients with tumor with MAGEC2 positive expression have a worse prognosis and a shorter metastasis free interval. Multivariate analyses showed that MAGEC2 expression was an independent risk factor for patient overall survival and metastasis-free survival. Breast cancer cells that overexpressed MAGEC2 had stronger migratory and invasive potential than control-treated cells. Epithelial markers (E-cadherin and cytokeratin) were down-regulated in MAGEC2-overexpressing cells compared to controls, whereas mesenchymal markers (vimentin and fibronectin) were upregulated. Our results indicate that MAGEC2 has a role in breast cancer metastasis through inducing epithelial-mesenchymal transition. In addition, MAGEC2 is a novel independent poor prognostic factor in patients with IDC. Thus, targeting MAGEC2 may provide a novel therapeutic strategy for

  20. Identification of mammary epithelial cells subject to chronic oxidative stress in mammary epithelium of young women and teenagers living in USA: Implication for breast carcinogenesis

    National Research Council Canada - National Science Library

    Weisz, Judith; Shearer, Debra A; Murata, Erin; Patrick, Susan D; Han, Bing; Berg, Arthur; Clawson, Gary A

    2012-01-01

    .... Breast reduction tissues from young women and teenagers, representative of the USA's high breast cancer incidence population, were studied using immunocytochemistry and a targeted PCR array in order...

  1. Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

    Science.gov (United States)

    Zhong, Aimei; Wang, Guohua; Yang, Jie; Xu, Qijun; Yuan, Quan; Yang, Yanqing; Xia, Yun; Guo, Ke; Horch, Raymund E; Sun, Jiaming

    2014-01-01

    True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co-culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non-macromastic epithelial cells when co-cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia-derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co-culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co-cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. PMID:24720804

  2. The Epithelial-to-Mesenchymal Transition in Breast Cancer: Focus on Basal-Like Carcinomas

    Directory of Open Access Journals (Sweden)

    Monica Fedele

    2017-09-01

    Full Text Available Breast cancer is a heterogeneous disease that is characterized by a high grade of cell plasticity arising from the contribution of a diverse range of factors. When combined, these factors allow a cancer cell to transition from an epithelial to a mesenchymal state through a process of dedifferentiation that confers stem-like features, including chemoresistance, as well as the capacity to migrate and invade. Understanding the complex events that lead to the acquisition of a mesenchymal phenotype will therefore help to design new therapies against metastatic breast cancer. Here, we recapitulate the main endogenous molecular signals involved in this process, and their cross-talk with paracrine factors. These signals and cross-talk include the extracellular matrix; the secretome of cancer-associated fibroblasts, macrophages, cancer stem cells, and cancer cells; and exosomes with their cargo of miRNAs. Finally, we highlight some of the more promising therapeutic perspectives based on counteracting the epithelial-to-mesenchymal transition in breast cancer cells.

  3. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  4. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Horzinek, M C; Rottier, P J

    1995-01-01

    Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

  5. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  6. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Twite, Nicolas [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Andrei, Graciela [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Kummert, Caroline [ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Donner, Catherine [Department of Obstetrics and Gynecology, Erasme Hospital, Route de Lennik 808, 1070 Brussels (Belgium); Perez-Morga, David [Laboratory of Molecular Parasitology, Institut de Biologie et Médecine Moléculaires, Université Libre de Bruxelles, Gosselies (Belgium); De Vos, Rita [Pathology Department, U.Z. Leuven, Minderbroedersstraat 12, Leuven (Belgium); Snoeck, Robert, E-mail: Robert.Snoeck@Rega.kuleuven.be [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Marchant, Arnaud, E-mail: arnaud.marchant@ulb.ac.be [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium)

    2014-07-15

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.

  7. The contribution of heavy metals in cigarette smoke condensate to malignant transformation of breast epithelial cells and in vivo initiation of neoplasia through induction of a PI3K–AKT–NFκB cascade

    Energy Technology Data Exchange (ETDEWEB)

    Mohapatra, Purusottam; Preet, Ranjan; Das, Dipon; Satapathy, Shakti Ranjan [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India); Siddharth, Sumit [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India); Department of Infection Biology, Institute of Life Science, Nalco Square, Bhubaneswar, Orissa 751021 (India); Choudhuri, Tathagata [Department of Infection Biology, Institute of Life Science, Nalco Square, Bhubaneswar, Orissa 751021 (India); Wyatt, Michael D. [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC (United States); Kundu, Chanakya Nath, E-mail: cnkundu@gmail.com [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India)

    2014-01-01

    Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011 μg Cd per cigarette equivalent, and Cd (0.0003 μg Cd/1 × 10{sup 7} cells) was also detected in the lysates from MCF-10A cells treated with 25 μg/mL CSC. In similar manner to CSC, CdCl{sub 2} treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K–AKT–NFκB protein expression. An increase in the expression of PI3K–AKT–NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl{sub 2} treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins. - Highlights: • Repeated exposure of CSC causes malignant transformation in MCF-10A. • MCF-10A-Tr cells showed a distinct

  8. Transcription profiles of non-immortalized breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Holland James F

    2006-04-01

    Full Text Available Abstract Background Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Methods Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs were used in addition to commercially-available normal breast epithelial cells (HMECs, established breast cancer cell lines (T-est and established normal breast cells (N-est. The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. Results According to Significance Analysis of Microarray (SAM and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p Conclusion The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research.

  9. Breast cancer stem cells

    Directory of Open Access Journals (Sweden)

    Thomas W Owens

    2013-08-01

    Full Text Available Cancer metastasis, resistance to therapies and disease recurrence are significant hurdles to successful treatment of breast cancer. Identifying mechanisms by which cancer spreads, survives treatment regimes and regenerates more aggressive tumours are critical to improving patient survival. Substantial evidence gathered over the last 10 years suggests that breast cancer progression and recurrence is supported by cancer stem cells (CSCs. Understanding how CSCs form and how they contribute to the pathology of breast cancer will greatly aid the pursuit of novel therapies targeted at eliminating these cells. This review will summarise what is currently known about the origins of breast CSCs, their role in disease progression and ways in which they may be targeted therapeutically.

  10. Expression of miR-21 and its targets (PTEN, PDCD4, TM1) in flat epithelial atypia of the breast in relation to ductal carcinoma in situ and invasive carcinoma

    NARCIS (Netherlands)

    Qi, Liqiang; Bart, Joost; Tan, Lu Ping; Platteel, Inge; van der Sluis, Tineke; Huitema, Sippie; Harms, Geert; Fu, Li; Hollema, Harry; van den Berg, Anke

    2009-01-01

    Background: Flat epithelial atypia (FEA) of the breast is characterised by a few layers of mildly atypical luminal epithelial cells. Genetic changes found in ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDC) are also found in FEA, albeit at a lower concentration. So far, miRNA

  11. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    Science.gov (United States)

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  12. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

    Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

    Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally,

  13. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  14. Protons sensitize epithelial cells to mesenchymal transition.

    Science.gov (United States)

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M; Pluth, Janice M; Cucinotta, Francis A

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1.

  15. Airway epithelial cell tolerance to Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Verghese Margrith W

    2005-04-01

    Full Text Available Abstract Background The respiratory tract epithelium is a critical environmental interface that regulates inflammation. In chronic infectious airway diseases, pathogens may permanently colonize normally sterile luminal environments. Host-pathogen interactions determine the intensity of inflammation and thus, rates of tissue injury. Although many cells become refractory to stimulation by pathogen products, it is unknown whether the airway epithelium becomes either tolerant or hypersensitive in the setting of chronic infection. Our goals were to characterize the response of well-differentiated primary human tracheobronchial epithelial cells to Pseudomonas aeruginosa, to understand whether repeated exposure induced tolerance and, if so, to explore the mechanism(s. Methods The apical surface of well-differentiated primary human tracheobronchial epithelial cell cultures was repetitively challenged with Pseudomonas aeruginosa culture filtrates or the bacterial media control. Toxicity, cytokine production, signal transduction events and specific effects of dominant negative forms of signaling molecules were examined. Additional experiments included using IL-1β and TNFα as challenge agents, and performing comparative studies with a novel airway epithelial cell line. Results An initial challenge of the apical surface of polarized human airway epithelial cells with Pseudomonas aeruginosa culture filtrates induced phosphorylation of IRAK1, JNK, p38, and ERK, caused degradation of IκBα, generation of NF-κB and AP-1 transcription factor activity, and resulted in IL-8 secretion, consistent with activation of the Toll-like receptor signal transduction pathway. These responses were strongly attenuated following a second Pseudomonas aeruginosa, or IL-1β, but not TNFα, challenge. Tolerance was associated with decreased IRAK1 protein content and kinase activity and dominant negative IRAK1 inhibited Pseudomonas aeruginosa -stimulated NF-κB transcriptional

  16. Intracellular localization of the extracellular matrix enzyme lysyl oxidase in polarized epithelial cells

    Science.gov (United States)

    Jansen, Matthias K.; Csiszar, Katalin

    2007-01-01

    Considerable evidence supports novel functions for lysyl oxidase (LOX) beyond its traditional role in initiating crosslinkages in collagen and elastin within the extracellular matrix. These novel roles are particularly relevant during the transition of malignant epithelial cells towards a migratory and invasive phenotype. However, knowledge on cellular and matrix functions of LOX has been generated almost exclusively in mesenchymal cell types. But it is becoming increasingly evident that these cell types are not adequate to address these novel and highly significant roles for LOX in epithelial tissues. In this initial report, we demonstrate that active LOX is expressed by polarized MDCK II kidney and MCF-10A breast epithelial cells. Furthermore, we show evidence for the presence of mature LOX in the cytoplasm and establish these cell lines as models for epithelial LOX studies. PMID:17074474

  17. LincRNA-ROR induces epithelial-to-mesenchymal transition and contributes to breast cancer tumorigenesis and metastasis.

    Science.gov (United States)

    Hou, P; Zhao, Y; Li, Z; Yao, R; Ma, M; Gao, Y; Zhao, L; Zhang, Y; Huang, B; Lu, J

    2014-06-12

    LncRNAs have critical roles in various biological processes ranging from embryonic development to human diseases, including cancer progression, although their detailed mechanistic functions remain illusive. The lncRNA linc-ROR has been shown to contribute to the maintenance of induced pluripotent stem cells and embryonic stem cells. In this study, we discovered that linc-ROR was upregulated in breast tumor samples, and ectopic overexpression of linc-ROR in immortalized human mammary epithelial cells induced an epithelial-to-mesenchymal transition (EMT) program. Moreover, we showed that linc-ROR enhanced breast cancer cell migration and invasion, which was accompanied by generation of stem cell properties. Contrarily, silencing of linc-ROR repressed breast tumor growth and lung metastasis in vivo. Mechanistically, our data revealed that linc-ROR was associated with miRNPs and functioned as a competing endogenous RNA to mi-205. Specifically, linc-ROR prevented the degradation of mir-205 target genes, including the EMT inducer ZEB2. Thus our results indicate that linc-ROR functions as an important regulator of EMT and can promote breast cancer progression and metastasis through regulation of miRNAs. Potentially, the findings of this study implicate the relevance of linc-ROR as a possible therapeutic target for aggressive and metastatic breast cancers.

  18. C-phycocyanin suppresses transforming growth factor-β1-induced epithelial mesenchymal transition in human epithelial cells.

    Science.gov (United States)

    Pattarayan, Dhamotharan; Rajarajan, Dheeran; Ayyanar, Sivanantham; Palanichamy, Rajaguru; Subbiah, Rajasekaran

    2017-06-01

    Epithelial mesenchymal transition (EMT) is a process through which epithelial cells undergo multiple biochemical changes, causing them to differentiate into a mesenchymal-cell phenotype. This process has been shown to contribute to the development of fibrotic diseases. C-phycocyanin (C-PC) is a phycobiliprotein extracted from Spirulina platensis. This study was done to investigate the effect of C-PC on transforming growth factor-β1 (TGF-β1)-induced EMT and an EMT associated proliferation in human epithelial cell lines. Human adenocarcinoma cell line, A549 and breast cancer cell line, MCF-7 were treated with TGF-β1, and EMT-related genes expression, cell proliferation and cell cycle arrest were examined. C-PC suppressed the EMT as assessed by reduced expression of vimentin, type-1-collagen and fibronectin, and increased E-cadherin expression in TGF-β1 treated cells. Further, TGF-β1 treatment induced cell cycle arrest in S and G2/M phase in A549 cells. However, TGF-β1-mediated cell cycle arrest was significantly reversed by combined treatment with C-PC. The overall data suggested that C-PC suppresses TGF- β1-induced EMT and warrants further in vivo studies for future evaluation of C-PC as a potential antifibrotic agent. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

  19. Myoepithelial cells: their origin and function in breast morphogenesis and neoplasia

    DEFF Research Database (Denmark)

    Gudjonsson, Thorarinn; Adriance, Melissa C; Sternlicht, Mark D

    2005-01-01

    The human breast epithelium is a branching ductal system composed of an inner layer of polarized luminal epithelial cells and an outer layer of myoepithelial cells that terminate in distally located terminal duct lobular units (TDLUs). While the luminal epithelial cell has received the most atten...

  20. Induction of PTEN-p53 crosstalk in mammary epithelial cells: A novel mechanism of breast cancer prevention by the dietary factor genistein

    Science.gov (United States)

    Consumption of soy foods either at an early age or for lifetime has been associated with reduced risk for developing breast cancer in humans and in animal models. However, this association continues to be controversial, and the precise mechanisms for protection remain elusive. Among the soy products...

  1. Sequential Salinomycin Treatment Results in Resistance Formation through Clonal Selection of Epithelial-Like Tumor Cells

    Directory of Open Access Journals (Sweden)

    Florian Kopp

    2014-12-01

    Full Text Available Acquiring therapy resistance is one of the major obstacles in the treatment of patients with cancer. The discovery of the cancer stem cell (CSC–specific drug salinomycin raised hope for improved treatment options by targeting therapy-refractory CSCs and mesenchymal cancer cells. However, the occurrence of an acquired salinomycin resistance in tumor cells remains elusive. To study the formation of salinomycin resistance, mesenchymal breast cancer cells were sequentially treated with salinomycin in an in vitro cell culture assay, and the resulting differences in gene expression and salinomycin susceptibility were analyzed. We demonstrated that long-term salinomycin treatment of mesenchymal cancer cells resulted in salinomycin-resistant cells with elevated levels of epithelial markers, such as E-cadherin and miR-200c, a decreased migratory capability, and a higher susceptibility to the classic chemotherapeutic drug doxorubicin. The formation of salinomycin resistance through the acquisition of epithelial traits was further validated by inducing mesenchymal-epithelial transition through an overexpression of miR-200c. The transition from a mesenchymal to a more epithelial-like phenotype of salinomycin-treated tumor cells was moreover confirmed in vivo, using syngeneic and, for the first time, transgenic mouse tumor models. These results suggest that the acquisition of salinomycin resistance through the clonal selection of epithelial-like cancer cells could become exploited for improved cancer therapies by antagonizing the tumor-progressive effects of epithelial-mesenchymal transition.

  2. Cells of Origin of Epithelial Ovarian Cancers

    Science.gov (United States)

    2015-09-01

    lethal malignancy of the female reproductive system, largely due to the fact that most EOCs are diagnosed only after the cancer has metastasized into the...Epithelial ovarian cancer (EOC) is the most lethal malignancy of the female reproductive system, largely due to the fact that most EOCs are diagnosed only...experience in ovary research (ovarian physiology , oogonial stem cells) to work on this project. We also ! 5! obtained approval of our animal

  3. Dissection of a stem cell hierarchy in the human breast

    DEFF Research Database (Denmark)

    Rubner Fridriksdottir, Agla Jael

    where senescence is bypassed. These findings identify an adult human breast ductal stem cell activity and the earliest descendants thereof. Further characterization of the ductal stem cell niche, especially in terms of possible interaction with surrounding stromal cells (Sigurdsson and Fridriksdottir et......The human breast is a unique organ in that it undergoes most of its development after birth under the control of systemic hormones. Throughout the reproductive life of women, the breast gland undergoes changes in morphogenesis as evidenced by continuous cell proliferation, differentiation...... and apoptosis during each menstrual cycle. These changes are most prominent during pregnancy, lactation and involution after breast feeding. These highly dynamic changes are thought to rely on the presence of a breast epithelial stem cell population (reviewed in (Fridriksdottir et al. 2005)). Nevertheless...

  4. Sinomenine hydrochloride inhibits breast cancer metastasis by attenuating inflammation-related epithelial-mesenchymal transition and cancer stemness.

    Science.gov (United States)

    Li, Xiao; Li, Pingping; Liu, Chao; Ren, Yu; Tang, Xiaojiang; Wang, Ke; He, Jianjun

    2017-02-21

    Sinomenine hydrochloride (SH) has been investigated for its anti-tumor growth effect. We have previously reported that SH inhibited breast cancer cell proliferation via MAPKs signaling. However, whether SH could inhibit tumor metastasis has not been fully explored. In this study, we found that SH suppressed the metastasis potential of breast cancer cells. The wound healing and transwell assays showed that SH inhibited the migration and invasion ability of both 4T1 and MDA-MB-231 breast cancer cells. The orthotopic mouse model of 4T1 and the experimental mouse model of MDA-MB-231-luc (MDA-MB-231 cell line expressing firefly luciferase) demonstrated that SH treatment inhibited breast cancer metastasis by inhibiting epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties without obvious hepatotoxicity and renal toxicity. We also found that SH decreased spleen volume and weight in both mouse models, especially in the 4T1 mouse model. IL-6, a strong inflammatory factor causing EMT, was remarkably reduced. Overall, this anti-metastasis effect of SH could be possibly caused by attenuating inflammatory reaction, which led to inhibition of EMT and CSC characteristics of breast cancer cells. This study, together with our previous one, provides more evidence of SH as a potential drug for breast cancer therapy.

  5. Increased mammogram-induced DNA damage in mammary epithelial cells aged in vitro.

    Directory of Open Access Journals (Sweden)

    Laia Hernández

    Full Text Available Concerned about the risks of mammography screening in the adult population, we analyzed the ability of human mammary epithelial cells to cope with mammogram-induced DNA damage. Our study shows that an X-ray dose of 20 mGy, which is the standard dose received by the breast surface per two-view mammogram X-ray exploration, induces increased frequencies of DNA double-strand breaks to in vitro aged-but not to young-human mammary epithelial cells. We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones. Our studies point to an inefficient damage response of aged cells to low-dose radiation, this being due to both delayed and incomplete mobilization of repair proteins to DNA strand breaks. This inefficient damage response is translated into an important delay in double-strand break disappearance and consequent accumulation of unrepaired DNA breaks. The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration. Since our experiments were carried out in primary epithelial cell cultures in which cells age at the same time as they undergo replication-dependent telomere shortening, we needed to determine the contribution of these two factors to their phenotype. In this paper, we report that the exogenous expression of human telomerase retrotranscriptase in late population doubling epithelial cells does not rescue its delayed repair phenotype. Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres. Our findings of long-lasting double strand breaks and incomplete DNA break repair in the in vitro aged epithelial cells are in line with the increased carcinogenic risks of radiation exposures at older ages revealed by epidemiologic studies.

  6. Increased Mammogram-Induced DNA Damage in Mammary Epithelial Cells Aged In Vitro

    Science.gov (United States)

    Hernández, Laia; Terradas, Mariona; Martín, Marta; Feijoo, Purificación; Soler, David; Tusell, Laura; Genescà, Anna

    2013-01-01

    Concerned about the risks of mammography screening in the adult population, we analyzed the ability of human mammary epithelial cells to cope with mammogram-induced DNA damage. Our study shows that an X-ray dose of 20 mGy, which is the standard dose received by the breast surface per two-view mammogram X-ray exploration, induces increased frequencies of DNA double-strand breaks to in vitro aged–but not to young–human mammary epithelial cells. We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones. Our studies point to an inefficient damage response of aged cells to low-dose radiation, this being due to both delayed and incomplete mobilization of repair proteins to DNA strand breaks. This inefficient damage response is translated into an important delay in double-strand break disappearance and consequent accumulation of unrepaired DNA breaks. The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration. Since our experiments were carried out in primary epithelial cell cultures in which cells age at the same time as they undergo replication-dependent telomere shortening, we needed to determine the contribution of these two factors to their phenotype. In this paper, we report that the exogenous expression of human telomerase retrotranscriptase in late population doubling epithelial cells does not rescue its delayed repair phenotype. Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres. Our findings of long-lasting double strand breaks and incomplete DNA break repair in the in vitro aged epithelial cells are in line with the increased carcinogenic risks of radiation exposures at older ages revealed by epidemiologic studies. PMID:23667571

  7. Oleanolic Acid, a Compound Present in Grapes and Olives, Protects against Genotoxicity in Human Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Cristina Sánchez-Quesada

    2015-07-01

    Full Text Available Oleanolic acid (AO and maslinic acid (MA are constituents of the skins of different fruits, including olives and white or red grapes. Although both compounds are known to have beneficial properties against different types of cancers, thus far, there are no studies about their chemopreventive effects in human breast cancer. Thus, we sought to elucidate whether both compounds possess chemopreventive activity. Two cell lines of human breast cancer cells and one noncancerous human mammary epithelial cells were used to determine the effects of OA and MA. The results showed that OA inhibited the proliferation and increased the oxidative stress of highly invasive cells. Additionally, OA decreased oxidative stress and oxidative damage to the DNA in human mammary epithelial cells. These results suggest that OA could act as a chemopreventive agent in human breast cancer and could inhibit the proliferation of highly invasive breast cancer cells.

  8. Lactobacillus decelerates cervical epithelial cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Katarina Vielfort

    Full Text Available We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

  9. MUCOUS CELLS IN THE EPITHELIAL LINING OF DENTIGEROUS CYST

    OpenAIRE

    Pratiksha; Sangeeta; Sumanta; Prashanti; Sathyajitraje; Rajkumar

    2014-01-01

    The epithelial lining of both the developmental and inflammatory cysts of odontogenic origin are primarily composed of squamous epithelium. Various forms of metaplasia and degenerations are observed in these epithelial linings e.g. mucous cells, ciliated cells, para and/or orthokeratinization and formation of hyaline bodies. The present study was designed to investigate the incidences of mucous cells in the epithelial lining of dentigerous cyst. Mucous cells were observed ...

  10. Human Milk Oligosaccharides Protect Bladder Epithelial Cells Against Uropathogenic Escherichia coli Invasion and Cytotoxicity

    Science.gov (United States)

    Lin, Ann E.; Autran, Chloe A.; Espanola, Sophia D.; Bode, Lars; Nizet, Victor

    2014-01-01

    The invasive pathogen uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). Recurrent infection that can progress to life-threatening renal failure has remained as a serious global health concern in infants. UPEC adheres to and invades bladder epithelial cells to establish infection. Studies have detected the presence of human milk oligosaccharides (HMOs) in urine of breast-fed, but not formula-fed, neonates. We investigated the mechanisms HMOs deploy to elicit protection in human bladder epithelial cells infected with UPEC CFT073, a prototypic urosepsis-associated strain. We found a significant reduction in UPEC internalization into HMO-pretreated epithelial cells without observing any significant effect in UPEC binding to these cells. This event coincides with a rapid decrease in host cell cytotoxicity, recognized by LIVE/DEAD staining and cell detachment, but independent of caspase-mediated or mitochondrial-mediated programmed cell death pathways. Further investigation revealed HMOs, and particularly the sialic acid-containing fraction, reduced UPEC-mediated MAPK and NF-κB activation. Collectively, our results indicate that HMOs can protect bladder epithelial cells from deleterious cytotoxic and proinflammatory effects of UPEC infection, and may be one contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast-fed infants. PMID:23990566

  11. Do myoepithelial cells hold the key for breast tumorprogression?

    Energy Technology Data Exchange (ETDEWEB)

    Polyak, Kornelia; Hu, Min

    2005-11-18

    Mammary myoepithelial cells have been the foster child of breast cancer biology and have been largely ignored since they were considered to be less important for tumorigenesis than luminal epithelial cells from which most of breast carcinomas are thought to arise. In recent years as our knowledge in stem cell biology and the cellular microenvironment has been increasing myoepithelial cells are slowly starting to gain more attention. Emerging data raise the hypothesis if myoepithelial cells play a key role in breast tumor progression by regulating the in situ to invasive carcinoma transition and if myoepithelial cells are part of the mammary stem cell niche. Paracrine interactions between myoepithelial and luminal epithelial cells are known to be important for cell cycle arrest, establishing epithelial cell polarity, and inhibiting migration and invasion. Based on these functions normal mammary myoepithelial cells have been called ''natural tumor suppressors''. However, during tumor progression myoepithelial cells seem to loose these properties and eventually they themselves diminish as tumors become invasive. Better understanding of myoepithelial cell function and their role in tumor progression may lead to their exploitation for cancer therapeutic and preventative measures.

  12. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  13. Aluminium chloride promotes anchorage-independent growth in human mammary epithelial cells

    OpenAIRE

    Sappino, André-Pascal; Buser Llinares, Raphaële; Lesne, Laurence; Gimelli, Stefania; Bena, Frédérique; Belin, Dominique; Mandriota, Stefano Jacopo

    2012-01-01

    Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspira...

  14. Cell reintegration: Stray epithelial cells make their way home.

    Science.gov (United States)

    Wilson, Tyler J; Bergstralh, Dan T

    2017-06-01

    Ongoing work shows that misplaced epithelial cells have the capacity to reintegrate back into tissue layers. This movement appears to underlie tissue stability and may also control aspects of tissue structure. A recent study reveals that cell reintegration in at least one tissue, the Drosophila follicular epithelium, is based on adhesion molecules that line lateral cell surfaces. In this article we will review these observations, discuss their implications for epithelial tissue development and maintenance, and identify future directions for study. © 2017 WILEY Periodicals, Inc.

  15. Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

    Directory of Open Access Journals (Sweden)

    Loredana Alberti

    Full Text Available Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites or CTCFL (CTCF-like is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1 and cancer stem cell markers (ABCG2, CD44 and ALDH1 genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7. Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

  16. Effects of epiplakin-knockdown in cultured corneal epithelial cells

    OpenAIRE

    Kokado, Masahide; Okada, Yuka; Miyamoto, Takeshi; Yamanaka, Osamu; Saika, Shizuya

    2016-01-01

    Background To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse. Methods Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and prolifer...

  17. Novel application of artificial dermis plus autologous vital epithelial cells: improved wound epithelialization.

    Science.gov (United States)

    Lee, Li-Tzu; Kwan, Po-Cheung; Wong, Yong-Kie

    2010-02-01

    The purpose of this study was to evaluate artificial dermis with the simultaneous addition of autologous epithelial cells for oral lesion defect reconstruction. Surgical wounds reconstructed with artificial dermis plus scraped epithelial cells were evaluated in 5 patients with oral benign lesions or squamous cell carcinoma. Clinical follow-up indices included scar formation and tissue surface texture observation. The neomucosal layers were analyzed histologically to establish the degree of epithelialization. Clinical observation showed that the oral mucosal texture was smoother in artificial dermis with added epithelial cells at 4 weeks postoperation compared with artificial dermis alone. The wound contraction and scar formation processes were slow. Viable epithelial cells with flat rete ridges remained in the artificial dermis, and a neoepithelial layer was present in the histological findings. We showed that healthy granulation tissue and neoepithelial formation in artificial dermis with epithelial cells was beneficial for the repair of oral defects. Scraping oral epithelial cells and applying them to artificial dermis assisted in the early preparation of composite grafts and minimized requirement for donor sites. This technique may improve the treatment of patients with oral benign tumors and early-stage squamous cell carcinoma. Copyright 2010 Elsevier. Published by Elsevier B.V. All rights reserved.

  18. Mammary Development and Breast Cancer: The Role of Stem Cells

    Science.gov (United States)

    Ercan, C.; van Diest, P.J.; Vooijs, M.

    2014-01-01

    The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer. Here we provide an overview on the functional and molecular identification of mammary stem cells in the context of both normal breast development and breast cancer. We discuss the contribution of some key signaling pathways with an emphasis on Notch receptor signaling, a cell fate determination pathway often deregulated in breast cancer. A further understanding of the biological roles of the Notch pathway in mammary stem cell behavior and carcinogenesis might be relevant for the development of future therapies. PMID:21506923

  19. Modulation of epithelial cell polarity by bacterial pathogens.

    Science.gov (United States)

    Tapia, Rocio; Kralicek, Sarah E; Hecht, Gail A

    2017-10-01

    Epithelial cells constitute a physical barrier that aids in protecting the host from microbial pathogens. Polarized epithelial cells contain distinct apical and basolateral membrane domains separated by intercellular junctions, including tight junctions (TJs), which contribute to the maintenance of apical-basal polarity. Polarity complexes also contribute to the establishment of TJ formation. Several pathogens perturb epithelial TJ barrier function and structure in addition to causing a loss of apical-basal polarity. Here, we review the impact of pathogenic bacteria on the disruption of cell-cell junctions and epithelial polarity. © 2017 New York Academy of Sciences.

  20. Palbociclib inhibits epithelial-mesenchymal transition and metastasis in breast cancer via c-Jun/COX-2 signaling pathway.

    Science.gov (United States)

    Qin, Ge; Xu, Fei; Qin, Tao; Zheng, Qiufan; Shi, Dingbo; Xia, Wen; Tian, Yun; Tang, Yanlai; Wang, Jingshu; Xiao, Xiangshen; Deng, Wuguo; Wang, Shusen

    2015-12-08

    Palbociclib, a highly selective CDK4/6 inhibitor, has been shown to be a novel anti-tumor agent that suppresses breast cancer cell proliferation. However, its anti-metastasis activity remains controversial. In the present study, we evaluated whether palbociclib prevented breast cancer cell metastasis and revealed its regulatory mechanism. We found that palbociclib inhibited migration and invasion in the breast cancer cells MDA-MB-231 and T47D. The epithelial-mesenchymal transition (EMT) markers, vimentin and Snail, were down-regulated with palbociclib treatment. Moreover, we revealed that this inhibition was mediated by the c-Jun/COX-2 pathway. COX-2 was decreased after palbociclib treatment. The production of PGE2 was also reduced along with COX-2. Additionally, our data showed that c-Jun, a crucial transcriptional regulator of COX-2, was down-regulated by palbociclib. We found that palbociclib weakened the COX-2 promoter binding activity of c-Jun and prevented its translocation from the cytoplasm to cell nuclei. Bioluminescence imaging and tail intravenous injection were used to evaluate the anti-metastasis effect of palbociclib in vivo. The data demonstrated that palbociclib reduced breast cancer metastasis to the lung. These results therefore demonstrated that the anti-metastasis activity of palbociclib is mediated via the c-Jun/COX-2 signaling pathway by inhibiting EMT in breast cancer cells.

  1. Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal

    OpenAIRE

    Gradilone, Angela; Raimondi, Cristina; Nicolazzo, Chiara; Petracca, Arianna; Gandini, Orietta; Vincenzi, Bruno; Naso, Giuseppe; Aglian?, Anna Maria; Cortesi, Enrico; Gazzaniga, Paola

    2011-01-01

    Abstract Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial ...

  2. Cyclophosphamide or Denileukin Diftitox Followed By Expanding a Patient's Own T Cells in the Laboratory in Treating Patients With HER-2/Neu Overexpressing Metastatic Breast Cancer, Ovarian Cancer, or Non-Small Cell Lung Cancer Previously Treated With HER-2/Neu Vaccine

    Science.gov (United States)

    2014-11-07

    HER2-positive Breast Cancer; Recurrent Breast Cancer; Recurrent Non-small Cell Lung Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Stage IV Breast Cancer; Stage IV Non-small Cell Lung Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor

  3. Clinicopathological study of rare invasive epithelial tumors of breast: An institutional study

    Directory of Open Access Journals (Sweden)

    Karthik Kasireddy

    2016-01-01

    Full Text Available Introduction: Invasive breast cancer (BC is the most common carcinoma in women. It accounts for 22% of all female cancers. Most tumors are derived from mammary duct epithelium, and up to 75% of BCs are ductal carcinomas. The second most common tumor is invasive lobular carcinoma. However, there are many variants which are less common but well defined by the World Health Organization classification. They comprise <10% of breast tumors. Their clinical behavior differs greatly. Hence, it is important to know their main histomorphological features to make the best treatment of choice and to foresee prognosis. Aims and Objectives: To study the histomorphological features, incidence, and clinical features of rare invasive epithelial tumors of the breast. Materials and Methods: This study was done in the department of pathology, Sri Devaraj Urs Medical College, Kolar. All the neoplastic breast lesions over a period of 5 years (July 2010-September 2015 are included in the study. Clinical features and other details (estrogen receptor/progesterone receptor, human epidermal receptor-2, lymph nodes are obtained from the department (surgery records. Specimens are received and preserved in 10% formalin and are subjected to routine histopathological processing. Hematoxylin and eosin sections are studied, and a morphological diagnosis is given. All rare invasive epithelial breast tumors will be reviewed meticulously. Results and Conclusion: A total number of invasive epithelial tumors of breast were 105. The most common presenting symptom was breast lump. Rare invasive epithelial breast tumors account to 28.5%. The age range from 15 to 70 years. Most common, rare invasive epithelial tumor in our study is medullary carcinoma. Hence, it is imperative to always maintain a Hawks vigil during microscopic diagnosis to know prognosis of the condition and to facilitate early and prompt treatment to the patient.

  4. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  5. Cancer Stem Cells and Epithelial Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Sheetal Dyall

    2010-01-01

    Full Text Available The cancer stem cell hypothesis is becoming more widely accepted as a model for carcinogenesis. Tumours are heterogeneous both at the molecular and cellular level, containing a small population of cells that possess highly tumourigenic “stem-cell” properties. Cancer stem cells (CSCs, or tumour-initiating cells, have the ability to self-renew, generate xenografts reminiscent of the primary tumour that they were derived from, and are chemoresistant. The characterisation of the CSC population within a tumour that drives its growth could provide novel target therapeutics against these cells specifically, eradicating the cancer completely. There have been several reports describing the isolation of putative cancer stem cell populations in several cancers; however, no defined set of markers has been identified that conclusively characterises “stem-like” cancer cells. This paper highlights the current experimental approaches that have been used in the field and discusses their limitations, with specific emphasis on the identification and characterisation of the CSC population in epithelial ovarian cancer.

  6. Hugl1 and Hugl2 in mammary epithelial cells: polarity, proliferation, and differentiation.

    Directory of Open Access Journals (Sweden)

    Atlantis Russ

    Full Text Available Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.

  7. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Labovsky Vivian

    2012-06-01

    Full Text Available Abstract Background While breast cancer (BC is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG, receptor activator of nuclear factor kappa B ligand (RANKL, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, stromal cell-derived factor-1 (SDF-1, and their receptors (R in 2 human BC cell lines, MDA-MB-231 and MCF-7. Methods OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses. Results MCF-7 cells released higher levels of OPG in conditioned media (CM than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. Conclusions MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

  8. The Contribution of the Airway Epithelial Cell to Host Defense

    OpenAIRE

    Frauke Stanke

    2015-01-01

    In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how t...

  9. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    Energy Technology Data Exchange (ETDEWEB)

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R. (Dana-Farber Cancer Institute, Boston, MA (USA))

    1990-01-01

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV.

  10. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  11. Intestinal transport: studies with isolated epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kimmich, G.A.

    1979-12-01

    Isolated intestinal epithelial cells have been extremely useful for characterizing the nature of intestinal absorption processes and for providing insight into the energetics of Na/sup +/-dependent transport systems. This report describes a number of experimental approaches which have been used for investigating the specific epithelial transport systems involved in sugar absorption, but provides information which ultimately should prove useful for characterizing a number of different intestinal transport events. Similar experiments should also prove useful for exploring the effect of environmental agents on the function of intestinal tissue. In the case of sugars, net absorption is accomplished via a mucosal, Na/sup +/-dependent concentrative transport system acting in sequence with a passive serosal system which does not require Na/sup +/. The serosal system limits the full gradient-forming capability of the muscosal system. Agents such as phloretin or cytochalasin B which inhibit serosal transport allow the cells to establish sugar gradients as high as 70 fold in contrast to 10 to 15 fold gradients observed for control cells. Sevety-fold sugar gradients cannot be explained in terms of the energy available in the electrochemical potential for Na/sup +/ if the Na/sub 2/: sugar coupling stoichiometry is 1:1 as commonly assumed. New information indicates that the true Na/sup +/:sugar stoichiometry is in fact 2:1. Flow of two Na/sup +/ ions per sugar molecule down the transmembrane electrochemical potential for Na/sup +/ provides more than sufficient energy to account for observed 70 fold sugar gradients. If flow of sugar by other routes could be completely inhibited, theoretical sugar gradients as high as 400 could be achieved assuming that the cells maintain a membrane potential of -36 mV as measured for intact tissue.

  12. Uranium induces apoptosis in lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Periyakaruppan, Adaikkappan; Sarkar, Shubhashish; Sadanandan, Bindu; Thomas, Renard; Wilson, Bobby L. [Texas Southern University, Environmental Toxicology Program, Department of Chemistry, Houston, TX (United States); Ravichandran, Prabakaran; Sharma, Chidananda S.; Ramesh, Vani; Hall, Joseph C.; Ramesh, Govindarajan T. [Norfolk State University, Molecular Toxicology Laboratory, Department of Biology, Center for Biotechnology and Biomedical Sciences, Norfolk, VA (United States)

    2009-06-15

    Uranium is a naturally occurring radioactive material present everywhere in the environment. It is toxic because of its chemical or radioactive properties. Uranium enters environment mainly from mines and industry and cause threat to human health by accumulating in lungs as a result of inhalation. In our previous study, we have shown the effectiveness of antioxidant system response to the oxidative stress induced by uranyl acetate (UA) in rat lung epithelial (LE) cells. As part of our continuing studies; here, we investigated the mechanism underlying when LE cells are exposed to different concentration of UA. Oxidative stress may lead to apoptotic signaling pathways. LE cells treated with 0.25, 0.5 and 1 mM of UA results in dose and time-dependent increase in activity of both caspases-3 and -8. Increase in the concentration of cytochrome-c oxidase in cytosol was seen in LE cells treated with 1 mM UA as a result of mitochondria membrane permeability. The cytochrome-c leakage may trigger the apoptotic pathway. TUNEL assay performed in LE cells treated with 1 mM of UA showed significant incorporation of dNTPs in the nucleus after 24 h. In the presence of the caspase inhibitors, we observed the significant decrease in the activity of caspases-8 and -3 in 0.5 and 1 mM UA-treated LE cells. (orig.)

  13. Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal

    Science.gov (United States)

    Gradilone, Angela; Raimondi, Cristina; Nicolazzo, Chiara; Petracca, Arianna; Gandini, Orietta; Vincenzi, Bruno; Naso, Giuseppe; Aglianò, Anna Maria; Cortesi, Enrico; Gazzaniga, Paola

    2011-01-01

    Abstract Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial mesenchymal transition. The gain of mesenchymal markers in CTC was correlated to prognosis of patients in a follow-up of 24 months. The presence of mesenchymal markers on CTC more accurately predicted worse prognosis than the expression of cytokeratins alone. Because of the frequent loss of epithelial antigens by CTC, assays targeting epithelial antigens may miss the most invasive cell population. Thus, there is an urgent need to improve detection methods to identify CTC which undergone epithelial mesenchymal transition program. PMID:21352474

  14. Nutrients released by gastric epithelial cells enhance Helicobacter pylori growth

    NARCIS (Netherlands)

    van Amsterdam, Karin; van der Ende, Arie

    2004-01-01

    Background. Helicobacter pylori survives and proliferates in the human gastric mucosa. In this niche, H. pylori adheres to the gastric epithelial cells near the tight junctions. In vitro, H. pylori proliferated well in tissue-culture medium near gastric epithelial cells. However, in the absence of

  15. In vitro effect of smokeless tobacco on gingival epithelial cells

    Directory of Open Access Journals (Sweden)

    Arzu Beklen

    2017-05-01

    Epithelial cells are the first line of defense against pathogens in the oral cavity. The results suggest that smokeless tobacco not only inhibits the growth of epithelial cells but also induce the generation of inflammatory cytokines which leads to smokeless tobacco-exacerbated disease.

  16. Genome-wide ChIP-seq analysis of human TOP2B occupancy in MCF7 breast cancer epithelial cells

    Directory of Open Access Journals (Sweden)

    Catriona M. Manville

    2015-11-01

    Full Text Available We report the whole genome ChIP seq for human TOP2B from MCF7 cells. Using three different peak calling methods, regions of binding were identified in the presence or absence of the nuclear hormone estradiol, as TOP2B has been reported to play a role in ligand-induced transcription. TOP2B peaks were found across the whole genome, 50% of the peaks fell either within a gene or within 5 kb of a transcription start site. TOP2B peaks coincident with gene promoters were less frequently associated with epigenetic features marking active promoters in estradiol treated than in untreated cells. Significantly enriched transcription factor motifs within the DNA sequences underlying the peaks were identified. These included SP1, KLF4, TFAP2A, MYF, REST, CTCF, ESR1 and ESR2. Gene ontology analysis of genes associated with TOP2B peaks found neuronal development terms including axonogenesis and axon guidance were significantly enriched. In the absence of functional TOP2B there are errors in axon guidance in the zebrafish eye. Specific heparin sulphate structures are involved in retinal axon targeting. The glycosaminoglycan biosynthesis–heparin sulphate/heparin pathway is significantly enriched in the TOP2B gene ontology analysis, suggesting changes in this pathway in the absence of TOP2B may cause the axon guidance faults.

  17. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling.

    Science.gov (United States)

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-07-19

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity.

  18. Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Ying Qu

    Full Text Available Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture, three-dimensional (3D "on-top" Matrigel, 3D "cell-embedded" Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

  19. Isolation of fibroblasts and epithelial cells in bronchoalveolar lavage (BAL).

    Science.gov (United States)

    Pollock, Kathryn; Albares, Luke; Wendt, Chris; Hubel, Allison

    2013-04-01

    The long-term outcome of lung transplants is poor with 60%-70% of patients developing chronic rejection. Chronic rejection is manifested histologically by obliterative bronchiolitis with bronchiolitis obliterans syndrome (BOS), the clinical surrogate. Recent studies suggest that fibroblasts and epithelial cells present in bronchoalveolar lavage (BAL) may be a clinically relevant biomarker for BOS. The goal of this investigation was to develop a fast, repeatable method to individually isolate these low-frequency cell types. Fibroblasts and epithelial cells were isolated from BAL using attachment methods and the phenotype of the cells confirmed using immunostaining for vimentin (fibroblasts) and epithelial cell adhesion molecule (EpCAM, epithelial cells). Both fibroblasts and epithelial cells were isolated in every sample of BAL processed with the frequency of fibroblasts ranging from 0.03% to 0.48% and epithelial cells ranging from 0.05% to 1.5% of the total sample. Additional studies were performed using cytospins of cells after macrophages were depleted; cells exhibiting characteristics of both fibroblasts and epithelial cells were observed. The frequency of the cells of interest suggests that conventional methods of immunomagnetic isolation will not be effective in isolating these subpopulations. Finally, some of the low-frequency cells isolated via cytospin exhibit characteristics of epithelial to mesenchymal transition (which was not observed in plating incubations), indicating that the epithelial to mesenchymal cell transition fibroblasts may be nonadherent. In future studies, this technique and dataset may be of use to statistically correlate low-frequency cell type abundance to the onset and development of BOS.

  20. Multi-functionality and plasticity characterize epithelial cells in Hydra

    Science.gov (United States)

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  1. MAP3K4/CBP Regulated H2B Acetylation Controls Epithelial-Mesenchymal Transition in Trophoblast Stem Cells

    Science.gov (United States)

    Abell, Amy N.; Jordan, Nicole Vincent; Huang, Weichun; Prat, Aleix; Midland, Alicia A.; Johnson, Nancy L.; Granger, Deborah A.; Mieczkowski, Piotr A.; Perou, Charles M.; Gomez, Shawn M.; Li, Leping; Johnson, Gary L.

    2011-01-01

    SUMMARY Epithelial stem cells self-renew while maintaining multipotency, but the dependence of stem cell properties on maintenance of the epithelial phenotype is unclear. We previously showed that trophoblast stem (TS) cells lacking the protein kinase MAP3K4 maintain properties of both stemness and epithelial-mesenchymal transition (EMT). Here, we show that MAP3K4 controls the activity of the histone acetyltransferase CBP, and that acetylation of histones H2A and H2B by CBP is required to maintain the epithelial phenotype. Combined loss of MAP3K4/CBP activity represses expression of epithelial genes and causes TS cells to undergo EMT while maintaining their self-renewal and multipotency properties. The expression profile of MAP3K4 deficient TS cells defines an H2B acetylation regulated gene signature that closely overlaps with that of human breast cancer cells. Taken together, our data define an epigenetic switch that maintains the epithelial phenotype in TS cells and reveal previously unrecognized genes potentially contributing to breast cancer. PMID:21549327

  2. The Impact of Epithelial-Stromal Interactions on Human Breast Tumor Heterogeneity

    Science.gov (United States)

    2015-10-01

    AWARD NUMBER: W81XWH-13-1-0357 TITLE: The Impact of Epithelial-Stromal Interactions on Human Breast Tumor Heterogeneity PRINCIPAL... Heterogeneity 5b. GRANT NUMBER W81XWH-13-1-0357 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Dr. Crista Thompson 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail... Heterogeneity is a key factor underlying the variability in patient response to treatment, especially in Triple-Negative (TN) breast cancer cases. In

  3. Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

    Science.gov (United States)

    Girard, Brian J; Regan Anderson, Tarah M; Welch, Siya Lem; Nicely, Julie; Seewaldt, Victoria L; Ostrander, Julie H

    2015-01-01

    Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

  4. Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

    Directory of Open Access Journals (Sweden)

    Brian J Girard

    Full Text Available Tamoxifen (Tam is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+ breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1 promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS mutant of PELP1 (PELP1-cyto were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

  5. DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation.

    Science.gov (United States)

    Faronato, Monica; Nguyen, Van T M; Patten, Darren K; Lombardo, Ylenia; Steel, Jennifer H; Patel, Naina; Woodley, Laura; Shousha, Sami; Pruneri, Giancarlo; Coombes, R Charles; Magnani, Luca

    2015-09-08

    The acquisition of endocrine therapy resistance in estrogen receptor α (ERα) breast cancer patients represents a major clinical problem. Notch signalling has been extensively linked to breast cancer especially in patients who fail to respond to endocrine therapy. Following activation, Notch intracellular domain is released and enters the nucleus where activates transcription of target genes. The numerous steps that cascade after activation of the receptor complicate using Notch as biomarker. Hence, this warrants the development of reliable indicators of Notch activity. DMXL2 is a novel regulator of Notch signalling not yet investigated in breast cancer. Here, we demonstrate that DMXL2 is overexpressed in a subset of endocrine therapy resistant breast cancer cell lines where it promotes epithelial to mesenchymal transition through hyper-activation of Notch signalling via V-ATPase dependent acidification. Following DMXL2 depletion or treatment with Bafilomycin A1, both EMT targets and Notch signalling pathway significantly decrease. We show for the first time that DMXL2 protein levels are significantly increased in ERα positive breast cancer patients that progress after endocrine therapy. Finally, we demonstrate that DMXL2 is a transmembrane protein with a potential extra-cellular domain. These findings identify DMXL2 as a novel, functional biomarker for ERα positive breast cancer.

  6. Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells

    OpenAIRE

    Mandriota, Stefano J.; Tenan, Mirna; Ferrari, Paolo; Sappino, Andr??Pascal

    2016-01-01

    Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti?acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long?term exposure to concentrations of a...

  7. Markers of fibrosis and epithelial to mesenchymal transition demonstrate field cancerization in histologically normal tissue adjacent to breast tumors

    Science.gov (United States)

    Trujillo, Kristina A.; Heaphy, Christopher M.; Mai, Minh; Vargas, Keith M.; Jones, Anna C.; Vo, Phung; Butler, Kimberly S.; Joste, Nancy E.; Bisoffi, Marco; Griffith, Jeffrey K

    2011-01-01

    Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT-PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient-matched, tumor-adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN-1 and TAHN-5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra-lobular fibroblasts expressing MMP2, SPARC, TGF-β3, which are inducers of EMT, were both prevalent in TAHN-1 tissues, sparse in TAHN-5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α-smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN-1 tissues. These results identify cellular processes that are differentially activated between TAHN-1 and TAHN-5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors. PMID:21105047

  8. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Science.gov (United States)

    Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

    2014-01-01

    Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  9. Epithelial cells as alternative human biomatrices for comet assay

    OpenAIRE

    Emilio eRojas; Yolanda eLorenzo; Kristiane eHuag-Berg; Bjørn eNicolaissen; Mahara eValverde

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many org...

  10. Epstein–Barr Virus Infection of Mammary Epithelial Cells Promotes Malignant Transformation

    Directory of Open Access Journals (Sweden)

    Hai Hu

    2016-07-01

    Full Text Available Whether the human tumor virus, Epstein–Barr Virus (EBV, promotes breast cancer remains controversial and a potential mechanism has remained elusive. Here we show that EBV can infect primary mammary epithelial cells (MECs that express the receptor CD21. EBV infection leads to the expansion of early MEC progenitor cells with a stem cell phenotype, activates MET signaling and enforces a differentiation block. When MECs were implanted as xenografts, EBV infection cooperated with activated Ras and accelerated the formation of breast cancer. Infection in EBV-related tumors was of a latency type II pattern, similar to nasopharyngeal carcinoma (NPC. A human gene expression signature for MECs infected with EBV, termed EBVness, was associated with high grade, estrogen-receptor-negative status, p53 mutation and poor survival. In 11/33 EBVness-positive tumors, EBV-DNA was detected by fluorescent in situ hybridization for the viral LMP1 and BXLF2 genes. In an analysis of the TCGA breast cancer data EBVness correlated with the presence of the APOBEC mutational signature. We conclude that a contribution of EBV to breast cancer etiology is plausible, through a mechanism in which EBV infection predisposes mammary epithelial cells to malignant transformation, but is no longer required once malignant transformation has occurred.

  11. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment......ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment...

  12. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment......ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment...

  13. A mouse model for Luminal epithelial like ER positive subtype of human breast cancer

    Directory of Open Access Journals (Sweden)

    Nagarajan P

    2007-09-01

    Full Text Available Abstract Background Generation of novel spontaneous ER positive mammary tumor animal model from heterozygous NIH nude mice. Methods Using brother-sister mating with pedigree expansion system, we derived a colony of heterozygous breeding females showing ER-Positive tumors around the age of 6 months. Complete blood picture, differential leukocyte count, and serum levels of Estrogen, Alanine amino transferase (SGPT, Aspartate amino transferase (SGOT, total protein and albumin were estimated. Aspiration biopsies and microbiology were carried out. Gross pathology of the tumors and their metastatic potential were assessed. The tumors were excised and further characterized using histopathology, cytology, electron microscopy (EM, molecular markers and Mouse mammary Tumor Virus – Long Terminal Repeats (MMTV LTR specific RT-PCR. Results The tumors originated from 2ndor 5thor both the mammary glands and were multi-nodulated with variable central necrosis accompanied with an accumulation of inflammatory exudate. Significant increases in estrogen, SGPT, SGOT and neutrophils levels were noticed. Histopathologically, invasive nodular masses of pleomorphic tubular neoplastic epithelial cells invaded fibro-vascular stroma, adjacent dermis and subcutaneous tissue. Metastatic spread through hematogenous and regional lymph nodes, into liver, lungs, spleen, heart and dermal lymphatics was observed. EM picture revealed no viral particles and MMTV-negativity was confirmed through MMTV LTR-specific RT-PCR. High expression of ER α, moderate to high expression of proliferating cell nuclear antigen (PCNA, moderate expression of vimentin and Cytokeratin 19 (K19 and low expression of p53 were observed in tumor sections, when compared with that of the normal mammary gland. Conclusion Since 75% of human breast cancer were classified ER-positive and as our model mimics (in most of the characteristics, such as histopathology, metastasis, high estrogen levels the ER

  14. Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells.

    Science.gov (United States)

    Mandriota, Stefano J; Tenan, Mirna; Ferrari, Paolo; Sappino, André-Pascal

    2016-12-15

    Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti-acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long-term exposure to concentrations of aluminium-in the form of aluminium chloride (AlCl3 )-in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl3 form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl3 . Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well-established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens. © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  15. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Leigh A Knodler

    Full Text Available Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV. We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1, but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

  16. Extra-virgin olive oil polyphenols inhibit HER2 (erbB-2)-induced malignant transformation in human breast epithelial cells: relationship between the chemical structures of extra-virgin olive oil secoiridoids and lignans and their inhibitory activities on the tyrosine kinase activity of HER2.

    Science.gov (United States)

    Menendez, Javier A; Vazquez-Martin, Alejandro; Oliveras-Ferraros, Cristina; Garcia-Villalba, Rocio; Carrasco-Pancorbo, Alegria; Fernandez-Gutierrez, Alberto; Segura-Carretero, Antonio

    2009-01-01

    Depending on their structure, some polyphenols (e.g. flavonoids) abundantly found in plant-derived beverages such as green tea can efficiently inhibit tyrosine kinase and serine/threonine kinase activities. Extra-virgin olive oil (EVOO - the juice of the olive obtained solely by pressing and consumed without any further refining process) is unique among other vegetable oils because of the high level of naturally occurring phenolic compounds. We explored the ability of EVOO polyphenols to modulate HER2 tyrosine kinase receptor-induced in vitro transformed phenotype in human breast epithelial cells. Using MCF10A normal breast epithelial cells retrovirally engineered to overexpress the wild-type sequence of human HER2, we further determined the relationship between chemical structures of EVOO-derived phenolics and their inhibitory activities on the tyrosine kinase activity of the HER2 oncoprotein. When the activation (phosphorylation) status of HER2 was semi-quantitatively measured the secoiridoids blocked HER2 signaling by rapidly reducing the activation status of the 1248 tyrosine residue (Y1248), the main autophosphorylation site of HER2. EVOO-derived single phenols tyrosol and hydroxytyrosol and the phenolic acid elenolic acid failed to significantly decrease HER2 tyrosine kinase activity. The anti-HER2 tyrosine kinase activity IC50 values were up to 5-times lower in the presence of EVOO-derived lignans and secoiridoids than in the presence of EVOO-derived single phenols and phenolic acids. EVOO polyphenols induced strong tumoricidal effects by selectively triggering high levels of apoptotic cell death in HER2-positive MCF10A/HER2 cells but not in MCF10A/pBABE matched control cells. EVOO lignans and secoiridoids prevented HER2-induced in vitro transformed phenotype as they inhibited colony formation of MCF10A/HER2 cells in soft-agar. Our current findings not only molecularly support recent epidemiological evidence revealing that EVOO-related anti-breast cancer

  17. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial

  18. Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

    NARCIS (Netherlands)

    P. Aparicio-Domingo (Patricia); M. Romera-Hernandez (Monica); J.J. Karrich (Julien J.); F.H.J. Cornelissen (Ferry); N. Papazian (Natalie); D.J. Lindenbergh-Kortleve (Dicky); J.A. Butler (James A.); L. Boon (Louis); M. Coles (Mark); J.N. Samsom (Janneke); T. Cupedo (Tom)

    2015-01-01

    textabstractDisruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence

  19. Aluminium chloride promotes anchorage-independent growth in human mammary epithelial cells.

    Science.gov (United States)

    Sappino, André-Pascal; Buser, Raphaële; Lesne, Laurence; Gimelli, Stefania; Béna, Frédérique; Belin, Dominique; Mandriota, Stefano J

    2012-03-01

    Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspirants, and in the range of those recently measured in the human breast, results in loss of contact inhibition and anchorage-independent growth. These effects were preceded by an increase of DNA synthesis, DNA double strand breaks (DSBs), and senescence in proliferating cultures. AlCl(3) also induced DSBs and senescence in proliferating primary human mammary epithelial cells. In contrast, it had no similar effects on human keratinocytes or fibroblasts, and was not detectably mutagenic in bacteria. MCF-10A cells morphologically transformed by long-term exposure to AlCl(3) display strong upregulation of the p53/p21(Waf1) pathway, a key mediator of growth arrest and senescence. These results suggest that aluminium is not generically mutagenic, but similar to an activated oncogene, it induces proliferation stress, DSBs and senescence in normal mammary epithelial cells; and that long-term exposure to AlCl(3) generates and selects for cells able to bypass p53/p21(Waf1) -mediated cellular senescence. Our observations do not formally identify aluminium as a breast carcinogen, but challenge the safety ascribed to its widespread use in underarm cosmetics. Copyright © 2012 John Wiley & Sons, Ltd.

  20. Epithelial cells as alternative human biomatrices for comet assay

    Directory of Open Access Journals (Sweden)

    Emilio eRojas

    2014-11-01

    Full Text Available The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes.Over a thirty year period, the comet assay in epithelial cells has been litlle employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  1. Epithelial cells as alternative human biomatrices for comet assay.

    Science.gov (United States)

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  2. Sodium selectivity of Reissner's membrane epithelial cells

    Directory of Open Access Journals (Sweden)

    Kim Kyunghee X

    2011-02-01

    Full Text Available Abstract Background Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC, which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196, RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3. By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. Conclusions These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala

  3. Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells

    NARCIS (Netherlands)

    Cereser, B. (Biancastella); M. Jansen (Marnix); Austin, E. (Emily); Elia, G. (George); Mcfarlane, T. (Taneisha); C.H.M. van Deurzen (Carolien); A.M. Sieuwerts (Anieta); M.G. Daidone (Maria Grazia); Tadrous, P.J. (Paul J); Wright, N.A. (Nicholas A); L. Jones (Louise); Mcdonald, S.A. (Stuart Ac)

    2017-01-01

    textabstractIt is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and

  4. Tissue architecture: the ultimate regulator of breast epithelial function

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, Mina J; Rizki, Aylin; Mian, Saira

    2003-10-20

    discuss the following: first, how our laboratory came to develop a model of the mammary gland acinus; second, what this model has told us about mechanisms that govern tissue specificity and malignancy; and third, possible directions for future studies. We summarize the evidence for the central role of ECM signaling in the maintenance of mammary function in culture and (more briefly) its role in tumorigenesis. This is followed by a discussion of the role that tissue architecture and tissue polarity (as opposed to cell polarity) may play in these processes. In an elegantly written and reasoned essay, Kirschner et al. coined the new science of developmental biology 'molecular vitalism'. They framed new concepts for self-organization as well as schemes for information flow in biological organization. Rao et al. reviewed and elaborated on differential-equation-based models of biochemical reaction networks and intracellular noise, with emphasis on bacteria and phage. Similarly, Hartwell et al. discussed the synergy between experiment and theory in elucidating 'modules' - collections of interacting molecules - and in unraveling how these modules collaborate to perform cellular functions such as signal transduction. We believe that many of these ideas will also be applicable to the maintenance of tissue specificity. As much as we agree with Kirschner et al. regarding the limitations of the machine analogy to biological systems, we conclude with thoughts on how we may proceed to model the complex tissue networks that govern breast tissue architecture. We suggest that our understanding of the structure and function of breast tissue would benefit from examining recent techniques for modeling large complex networks such as the World Wide Web and the Internet backbone among others.

  5. Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis : Caveolin-1 Antagonizes Cyclin D1 Function in Mammary Epithelial Cells

    OpenAIRE

    Williams, Terence M.; Sotgia, Federica; Lee, Hyangkyu; Hassan, Ghada; Di Vizio, Dolores; Bonuccelli, Gloria; Capozza, Franco; Mercier, Isabelle; Rui, Hallgeir; Pestell, Richard G.; Lisanti, Michael P.

    2006-01-01

    Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system ...

  6. Increased COX-2 expression in epithelial and stromal cells of high mammographic density tissues and in a xenograft model of mammographic density.

    Science.gov (United States)

    Chew, G L; Huo, C W; Huang, D; Hill, P; Cawson, J; Frazer, H; Hopper, J L; Haviv, I; Henderson, M A; Britt, K; Thompson, E W

    2015-08-01

    Mammographic density (MD) adjusted for age and body mass index is one of the strongest known risk factors for breast cancer. Given the high attributable risk of MD for breast cancer, chemoprevention with a safe and available agent that reduces MD and breast cancer risk would be beneficial. Cox-2 has been implicated in MD-related breast cancer risk, and was increased in stromal cells in high MD tissues in one study. Our study assessed differential Cox-2 expression in epithelial and stromal cells in paired samples of high and low MD human breast tissue, and in a validated xenograft biochamber model of MD. We also examined the effects of endocrine treatment upon Cox-2 expression in high and low MD tissues in the MD xenograft model. Paired high and low MD human breast tissue samples were immunostained for Cox-2, then assessed for differential expression and staining intensity in epithelial and stromal cells. High and low MD human breast tissues were separately maintained in biochambers in mice treated with Tamoxifen, oestrogen or placebo implants, then assessed for percentage Cox-2 staining in epithelial and stromal cells. Percentage Cox-2 staining was greater for both epithelial (p = 0.01) and stromal cells (p tissues. In high MD biochamber tissues, percentage Cox-2 staining was greater in stromal cells of oestrogen-treated versus placebo-treated tissues (p = 0.05).

  7. Regulated Mucin Secretion from Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Kenneth Bruce Adler

    2013-09-01

    Full Text Available Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3x10^6 D per monomer whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ~1 um in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among MARCKS, cysteine string protein (CSP, HSP70 and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG. Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the

  8. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

    OpenAIRE

    Bergstralh, Dan T.; Lovegrove, Holly?E.; St Johnston, Daniel

    2015-01-01

    This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb3248 Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epi...

  9. Proximal location of mouse prostate epithelial stem cells

    OpenAIRE

    Tsujimura, Akira; Koikawa, Yasuhiro; Salm, Sarah; Takao, Tetsuya; Coetzee, Sandra; Moscatelli, David; Shapiro, Ellen; Lepor, Herbert; Sun, Tung-Tien; Wilson, E. Lynette

    2002-01-01

    Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propos...

  10. SWCNTs induced autophagic cell death in human bronchial epithelial cells.

    Science.gov (United States)

    Park, Eun-Jung; Zahari, Nur Elida M; Lee, Eun-Woo; Song, Jaewhan; Lee, Jae-Hyeok; Cho, Myung-Haing; Kim, Jae-Ho

    2014-04-01

    Carbon nanotubes are being actively introduced in electronics, computer science, aerospace, and other industries. Thus, the urgent need for toxicological studies on CNTs is mounting. In this study, we investigated the alterations in cellular response with morphological changes induced by single-walled carbon nanotubes (SWCNTs) in BEAS-2B cells, a human bronchial epithelial cell line. At 24h after exposure, SWCNTs rapidly decreased ATP production and cell viability as well a slight increase in the number of cells in the subG1 and G1 phases. In addition, SWCNTs increased the expression of superoxide dismutase (SOD)-1, but not SOD-2, and the number of cells generating ROS. The concentration of Cu and Zn ions also increased in a dose-dependent manner in cells exposed to SWCNTs. SWCNTs significantly enhanced the release of nitric oxide, interleukin (IL)-6, and IL-8 and up-regulated the expression of chemokine- and cytokine-related genes. Furthermore, the levels of autophagy-related genes, especially the DRAM1 gene, and the autophagosome formation-related proteins, were clearly up-regulated together with an increase of autophagosome-like vacuoles. Based on these results, we suggest that SWCNTs induce autophagic cell death through mitochondrial dysfunction and cytosolic damage in human bronchial epithelial cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. PAF-Wnt signaling-induced cell plasticity is required for maintenance of breast cancer cell stemness.

    Science.gov (United States)

    Wang, Xin; Jung, Youn-Sang; Jun, Sohee; Lee, Sunhye; Wang, Wenqi; Schneider, Andrea; Sun Oh, Young; Lin, Steven H; Park, Bum-Joon; Chen, Junjie; Keyomarsi, Khandan; Park, Jae-Il

    2016-02-04

    Cancer stem cells (CSCs) contribute to tumour heterogeneity, therapy resistance and metastasis. However, the regulatory mechanisms of cancer cell stemness remain elusive. Here we identify PCNA-associated factor (PAF) as a key molecule that controls cancer cell stemness. PAF is highly expressed in breast cancer cells but not in mammary epithelial cells (MECs). In MECs, ectopic expression of PAF induces anchorage-independent cell growth and breast CSC marker expression. In mouse models, conditional PAF expression induces mammary ductal hyperplasia. Moreover, PAF expression endows MECs with a self-renewing capacity and cell heterogeneity generation via Wnt signalling. Conversely, ablation of endogenous PAF induces the loss of breast cancer cell stemness. Further cancer drug repurposing approaches reveal that NVP-AUY922 downregulates PAF and decreases breast cancer cell stemness. Our results unveil an unsuspected role of the PAF-Wnt signalling axis in modulating cell plasticity, which is required for the maintenance of breast cancer cell stemness.

  12. The Contribution of the Airway Epithelial Cell to Host Defense.

    Science.gov (United States)

    Stanke, Frauke

    2015-01-01

    In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how the epithelial cell senses the presence of pathogens and inflammatory conditions, which, in turn, facilitates the activation of CFTR and thus directly promotes pathogens clearance and innate immune defense on the surface of the epithelial cell. This paper summarizes functional data that describes the effect of cytokines, chemokines, infectious agents, and inflammatory conditions on the ion transport properties of the epithelial cell and relates these key properties to the molecular pathology of cystic fibrosis. Recent findings on the role of cystic fibrosis modifier genes that underscore the role of the epithelial ion transport in host defense and inflammation are discussed.

  13. The Contribution of the Airway Epithelial Cell to Host Defense

    Directory of Open Access Journals (Sweden)

    Frauke Stanke

    2015-01-01

    Full Text Available In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how the epithelial cell senses the presence of pathogens and inflammatory conditions, which, in turn, facilitates the activation of CFTR and thus directly promotes pathogens clearance and innate immune defense on the surface of the epithelial cell. This paper summarizes functional data that describes the effect of cytokines, chemokines, infectious agents, and inflammatory conditions on the ion transport properties of the epithelial cell and relates these key properties to the molecular pathology of cystic fibrosis. Recent findings on the role of cystic fibrosis modifier genes that underscore the role of the epithelial ion transport in host defense and inflammation are discussed.

  14. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    Science.gov (United States)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  15. Paradoxical antiproliferative effect by a murine mammary tumor-derived epithelial cell line

    Directory of Open Access Journals (Sweden)

    Scharovsky O Graciela

    2007-10-01

    Full Text Available Abstract Background Despite significant advancement in breast cancer therapy, there is a great need for a better understanding of the mechanisms involved in breast carcinogenesis and progression, as well as of the role of epigenetic contributions from stromal cells in mammary tumorigenesis. In this study, we isolated and characterized murine mammary tumor-derived epithelial and myofibroblast cell lines, and investigated the in vitro and in vivo effect of cellular soluble factors produced by the epithelial cell line on tumor cells. Methods Morphology, immunophenotype, cytogenetics, invasiveness, and tumorigenicity of epithelial (LM-234ep and myofibroblast (LM-234mf cell lines isolated from two murine mammary adenocarcinomas with common ancestor were studied. The in vitro effects of LM-234ep conditioned medium on proliferation, cell cycle distribution, and expression of cell cycle proteins, were investigated in LM-234mf cells, mouse melanoma cells (B16-F10, and human cervical adenocarcinoma cells (HeLa. The in vivo anti-tumor activity of LM-234ep conditioned media was evaluated in subcutaneous tumors formed in nude mice by B16-F10 and HeLa cells. Results LM-234ep cells were found to be cytokeratin positive and hipertriploid, whereas LM-234mf cells were α-smooth muscle actin positive and hypohexaploid. Chromosome aberrations were found in both cases. Only LM-234mf revealed to be invasive in vitro and to secrete active MMP-2, though neither of the cell types were able to produce progressing tumors. LM-234ep-derived factors were able to inhibit the in vitro growth of LM-234mf, B16-F10, and HeLa cells, inducing cell cycle arrest in G0/G1 phase. The administration of LM-234ep conditioned medium inhibited the growth of B16-F10 and HeLa tumors in nude mice. Conclusion Our data suggest the existence of epithelial cell variants with tumor suppressive properties within mammary tumors. To our knowledge, this is the first report showing antiproliferative and

  16. FGFR signaling maintains a drug persistent cell population following epithelial-mesenchymal transition

    Science.gov (United States)

    Brown, Wells S.; Akhand, Saeed Salehin; Wendt, Michael K.

    2016-01-01

    An emerging characteristic of drug resistance in cancer is the induction of epithelial-mesenchymal transition (EMT). However, the mechanisms of EMT-mediated drug resistance remain poorly defined. Therefore, we conducted long-term treatments of human epidermal growth factor receptor-2 (Her2)-transformed breast cancer cells with either the EGFR/Her2 kinase inhibitor, Lapatinib or TGF-β, a known physiological inducer of EMT. Both of these treatment regimes resulted in robust EMT phenotypes, but upon withdrawal a subpopulation of TGF-β induced cells readily underwent mesenchymal-epithelial transition, where as Lapatinib-induced cells failed to reestablish an epithelial population. The mesenchymal population that remained following TGF-β stimulation and withdrawal was quickly selected for during subsequent Lapatinib treatment, manifesting in inherent drug resistance. The Nanostring cancer progression gene panel revealed a dramatic upregulation of fibroblast growth factor receptor 1 (FGFR1) and its cognate ligand FGF2 in both acquired and inherent resistance. Mechanistically, FGF:Erk1/2 signaling functions to stabilize the EMT transcription factor Twist and thus maintain the mesenchymal and drug resistant phenotype. Finally, Lapatinib resistant cells could be readily eliminated using recently characterized covalent inhibitors of FGFR. Overall our data demonstrate that next-generation targeting of FGFR can be used in combination with Her2-targeted therapies to overcome resistance in this breast cancer subtype. PMID:27825137

  17. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

    Directory of Open Access Journals (Sweden)

    Wen-Yang Hu

    2017-08-01

    Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

  18. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Malizia, Andrea P.; Lacey, Noreen [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland); Walls, Dermot [School of Biotechnology, Dublin City University. Dublin, 9. Ireland (Ireland); Egan, Jim J. [Advanced Lung Disease and Lung Transplant Program, Mater Misericordiae University Hospital. 44, Eccles Street. Dublin, 7. Ireland (Ireland); Doran, Peter P., E-mail: peter.doran@ucd.ie [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland)

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  19. Proteoglycan synthesis and Golgi organization in polarized epithelial cells.

    Science.gov (United States)

    Dick, Gunnar; Akslen-Hoel, Linn K; Grøndahl, Frøy; Kjos, Ingrid; Prydz, Kristian

    2012-12-01

    A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.

  20. Human amniotic epithelial cells inhibit growth of epithelial ovarian cancer cells via TGF‑β1-mediated cell cycle arrest.

    Science.gov (United States)

    Bu, Shixia; Zhang, Qiuwan; Wang, Qian; Lai, Dongmei

    2017-11-01

    It is reported that human amniotic epithelial cells (hAECs) endow intrinsic antitumor effects on certain kinds of cancer. This research was designed to evaluate whether hAECs endowed potential anticancer properties on epithelial ovarian cancer (EOC) cells in vivo and in vitro, which has not been reported before. In this study, we established a xenografted BALB/c nude mouse model by subcutaneously co-injecting ovarian cancer cell line, SK-OV-3, and hAECs for 28 days. In ex vivo experiments, CCK‑8 cell viability assay, real-time PCR, cell counting assay, cell cycle analysis and immunohistochemistry (IHC) assay were used to detect the effects of hAEC‑secreted factors on the proliferation and cell cycle progression of EOC cells. A cytokine array was conducted to detect anticancer-related cytokines released from hAECs. Human recombinant TGF‑β1 and TGF‑β1 antibody were used to treat EOC cells and analyzed whether TGF‑β1 contributed to the cell cycle arrest. Results from in vivo and ex vivo experiments showed that hAEC-secreted factors and rhTGF‑β1 decreased proliferation of EOC cells and induced G0/G1 cell cycle arrest in cancer cells, which could be partially reversed by excess TGF‑β1 antibody. These data indicate that hAECs endow potential anticancer properties on epithelial ovarian cancer in vivo and in vitro which is partially mediated by hAEC‑secreted TGF‑β1-induced cell cycle arrest. This study suggests a potential application of hAEC‑based therapy against epithelial ovarian cancer.

  1. Preferential accumulation of 5-aminolevulinic acid-induced protoporphyrin IX in breast cancer: a comprehensive study on six breast cell lines with varying phenotypes

    Science.gov (United States)

    Millon, Stacy R.; Ostrander, Julie H.; Yazdanfar, Siavash; Brown, J. Quincy; Bender, Janelle E.; Rajeha, Anita; Ramanujam, Nirmala

    2010-01-01

    We describe the potential of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence as a source of contrast for margin detection in commonly diagnosed breast cancer subtypes. Fluorescence intensity of PpIX in untreated and ALA-treated normal mammary epithelial and breast cancer cell lines of varying estrogen receptor expression were quantitatively imaged with confocal microscopy. Percentage change in fluorescence intensity integrated over 610-700 nm (attributed to PpIX) of posttreated compared to pretreated cells showed statistically significant differences between four breast cancer and two normal mammary epithelial cell lines. However, a direct comparison of post-treatment PpIX fluorescence intensities showed no differences between breast cancer and normal mammary epithelial cell lines due to confounding effects by endogenous fluorescence from flavin adenine dinucleotide (FAD). Clinically, it is impractical to obtain pre- and post-treatment images. Thus, spectral imaging was demonstrated as a means to remove the effects of endogenous FAD fluorescence allowing for discrimination between post-treatment PpIX fluorescence of four breast cancer and two normal mammary epithelial cell lines. Fluorescence spectral imaging of ALA-treated breast cancer cells showed preferential PpIX accumulation regardless of malignant phenotype and suggests a useful contrast mechanism for discrimination of residual cancer at the surface of breast tumor margins.

  2. Cell lineage determinants as regulators of breast cancer metastasis.

    Science.gov (United States)

    Lu, Wei; Kang, Yibin

    2016-12-01

    The mammary epithelium is organized in a hierarchy of mammary stem cells (MaSCs), progenitors, and differentiated cells. The development and homeostasis of mammary gland are tightly controlled by a complex network of cell lineage regulators. These determinants of cellular hierarchy are frequently deregulated in breast tumor cells and closely associated with cancer progression and metastasis. They also contribute to the diversity of breast cancer subtypes and their distinct metastatic patterns. Cell fate regulators that normally promote stem/progenitor activities can serve as drivers for epithelial-mesenchymal transition and metastasis whereas regulators that promote terminal differentiation generally suppress metastasis. In this review, we discuss how some of the key factors function in normal mammary lineage determination and how these processes are hijacked by tumor cells to enhance metastasis. Understanding the molecular connections between normal development and cancer metastasis will enable the development of more specific and effective therapeutic approaches targeting metastatic tumor cells.

  3. The cell surface mucin podocalyxin regulates collective breast tumor budding.

    Science.gov (United States)

    Graves, Marcia L; Cipollone, Jane A; Austin, Pamela; Bell, Erin M; Nielsen, Julie S; Gilks, C Blake; McNagny, Kelly M; Roskelley, Calvin D

    2016-01-22

    Overexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression. MCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. Forced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary

  4. Cell volume regulation in epithelial physiology and cancer

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Hoffmann, Else Kay; Novak, Ivana

    2013-01-01

    regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes...... such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered...

  5. Formation of reactive oxygen species in rat epithelial cells upon ...

    Indian Academy of Sciences (India)

    Unknown

    2.1 Cell culture. RLE-6TN epithelial cells (ATCC Rockville, MD, USA) have been immortalized by transfection of rat alveolar type II cells with SV40 DNA (Driscoll et al 1995). Cells were maintained in Dulbecco's Modified .... candidate because it is bound to desferrioxamine with a stability constant near 1031. The hint that ...

  6. Metformin inhibits the proliferation of benign prostatic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Zongwei Wang

    Full Text Available Benign prostatic hyperplasia (BPH is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1 promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R and regulating cell cycle.BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA.Metformin (0.5-10mM, 6-48h significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the expression of IGF-1R

  7. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  8. Transforming growth factor-β-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression

    National Research Council Canada - National Science Library

    Wendt, M K; Smith, J A; Schiemann, W P

    2010-01-01

    Transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) have critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-β...

  9. Gene Expression Profiles Suggest Iron Transport Pathway in the Lactating Human Epithelial Cell.

    Science.gov (United States)

    Cai, Chenxi; Eck, Peter; Friel, James K

    2017-03-01

    The molecular background of iron excretion into breast milk has not been determined in humans. We determined the expression of known iron transporters in mRNA extracted from human milk fat globules to deduce which known transporters are responsible for iron excretion into human milk. The expression of iron transporters in mRNA from human milk fat globules and mouse mammary epithelial cell lines was determined by quantitative real-time polymerase chain reaction. The expression of the transferrin receptor 1 (TFRC), divalent metal transporter 1 (SLC11A2), transferrin (TF), and lactoferrin (LTF) was confirmed in RNA isolated from the human milk fat globule. Similar expression was observed in the mouse mammary epithelial cell line HC11 in resting and lactating phenotypes. No iron export protein could be determined in the RNA isolated from fat globules in human breast milk and a human mammary epithelial cell line. The lack of iron exporters in the human mammary epithelia, in conjunction with the presence of lactoferrin suggests that transmembrane transport is not a major route of iron excretion into human milk.

  10. Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration.

    Science.gov (United States)

    Kumar, J Dinesh; Steele, Islay; Moore, Andrew R; Murugesan, Senthil V; Rakonczay, Zoltan; Venglovecz, Viktoria; Pritchard, D Mark; Dimaline, Rodney; Tiszlavicz, Laszlo; Varro, Andrea; Dockray, Graham J

    2015-07-15

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling. Copyright © 2015 the American Physiological Society.

  11. Characterisation of cell adhesion in airway epithelial cell types using electric cell-substrate impedance sensing

    NARCIS (Netherlands)

    Heijink, I H; Brandenburg, S M; Noordhoek, J A; Postma, D S; Slebos, D-J; van Oosterhout, A J M

    Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell-substrate impedance

  12. From cells to tissue: A continuum model of epithelial mechanics

    Science.gov (United States)

    Ishihara, Shuji; Marcq, Philippe; Sugimura, Kaoru

    2017-08-01

    A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-level mechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellular rearrangements. This model incorporates stress and deformation tensors, which can be compared with experimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidated dynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, including a mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. This study provides an integrated scheme for the understanding of the orchestration of morphogenetic processes in individual cells to achieve epithelial tissue morphogenesis.

  13. In Vitro Compatibility of Contact Lenses With Corneal Epithelial Cells.

    Science.gov (United States)

    Vijay, Ajay K; Fadli, Zohra; Lakkis, Carol; Coles-Brennan, Chantal; Willcox, Mark D P

    2017-07-18

    To determine the interaction of contact lenses of different materials with corneal epithelial cells grown in tissue culture. Two different corneal epithelial cell lines were grown to confluence in culture media. Two hydrogel contact lenses with and without polyvinylpyrrolidone (PVP) {1-DAY ACUVUE MOIST (1-Day ACUVUE [hydrogel lenses]) and a silicone hydrogel contact lens, AIR OPTIX NIGHT & DAY} were removed from their blister packs, washed in phosphate-buffered saline, and applied to the cells. After exposure for 24 hr at 37°C, lenses were removed, and the corneal cells and supernatants processed. Supernatants from the cell assays were used to quantify the amount of 17 different cytokines that were produced using a multiplex bead assay. Cells were stained to assess amount of cell death (apoptosis or necrosis) or stained to determine the level of mitochondrial activity. Stimulants of necrotic death (latex) or apoptotic death (sorbitol) were used as positive controls. Cells produced cytokines during normal growth. Exposure of cells to the hydrogel lenses resulted in only minimal changes to normal production of cytokines, but latex or sorbitol produced the most change. Exposure of the cells to all three lenses caused 4% to 23% reduction in mitochondrial activity, whereas exposure to the positive controls caused 71% to 98% reduction in mitochondrial activity. Exposure of the corneal epithelial cells to contact lenses produced minimal morphological changes, whereas exposure to latex or sorbitol produced significant changes to the human corneal epithelial cell line. Exposure of corneal epithelial cells to contact lenses had minimal impact on their physiology. There was no difference in epithelial cell responses to hydrogel with or without PVP compared with the silicone hydrogel contact lens.

  14. Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

    Energy Technology Data Exchange (ETDEWEB)

    Lasalvia, Maria [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Castellani, Stefano [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); D’Antonio, Palma [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Perna, Giuseppe [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Carbone, Annalucia [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); Colia, Anna Laura; Maffione, Angela Bruna [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Capozzi, Vito [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Conese, Massimo, E-mail: massimo.conese@unifg.it [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy)

    2016-10-15

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity in

  15. Probiotics promote endocytic allergen degradation in gut epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Song, Chun-Hua [Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou (China); Liu, Zhi-Qiang [Department of Gastroenterology, The Second Hospital, Zhengzhou University, Zhengzhou (China); Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada); Huang, Shelly [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada); Zheng, Peng-Yuan, E-mail: medp7123@126.com [Department of Gastroenterology, The Second Hospital, Zhengzhou University, Zhengzhou (China); Yang, Ping-Chang, E-mail: yangp@mcmaster.ca [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  16. Mammary Adipose Tissue-Derived Lysophospholipids Promote Estrogen Receptor-Negative Mammary Epithelial Cell Proliferation.

    Science.gov (United States)

    Volden, Paul A; Skor, Maxwell N; Johnson, Marianna B; Singh, Puneet; Patel, Feenalie N; McClintock, Martha K; Brady, Matthew J; Conzen, Suzanne D

    2016-05-01

    Lysophosphatidic acid (LPA), acting in an autocrine or paracrine fashion through G protein-coupled receptors, has been implicated in many physiologic and pathologic processes, including cancer. LPA is converted from lysophosphatidylcholine (LPC) by the secreted phospholipase autotaxin (ATX). Although various cell types can produce ATX, adipocyte-derived ATX is believed to be the major source of circulating ATX and also to be the major regulator of plasma LPA levels. In addition to ATX, adipocytes secrete numerous other factors (adipokines); although several adipokines have been implicated in breast cancer biology, the contribution of mammary adipose tissue-derived LPC/ATX/LPA (LPA axis) signaling to breast cancer is poorly understood. Using murine mammary fat-conditioned medium, we investigated the contribution of LPA signaling to mammary epithelial cancer cell biology and identified LPA signaling as a significant contributor to the oncogenic effects of the mammary adipose tissue secretome. To interrogate the role of mammary fat in the LPA axis during breast cancer progression, we exposed mammary adipose tissue to secreted factors from estrogen receptor-negative mammary epithelial cell lines and monitored changes in the mammary fat pad LPA axis. Our data indicate that bidirectional interactions between mammary cancer cells and mammary adipocytes alter the local LPA axis and increase ATX expression in the mammary fat pad during breast cancer progression. Thus, the LPC/ATX/LPA axis may be a useful target for prevention in patients at risk of ER-negative breast cancer. Cancer Prev Res; 9(5); 367-78. ©2016 AACR. ©2016 American Association for Cancer Research.

  17. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

    Science.gov (United States)

    Bergstralh, Dan T; Lovegrove, Holly E; St Johnston, Daniel

    2015-11-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions.

  18. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  19. Effects of epiplakin-knockdown in cultured corneal epithelial cells.

    Science.gov (United States)

    Kokado, Masahide; Okada, Yuka; Miyamoto, Takeshi; Yamanaka, Osamu; Saika, Shizuya

    2016-05-20

    To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse. Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and proliferation were examined by using scratch assay and Alamar blue assay, respectively. Scratch assay and Alamar blue assay showed migration and proliferation of the cells was accelerated by epiplakin knockdown. siRNA-knockdown of epiplakin suppressed protein expression of E-cadherin, keratin 6 and vimentin. Decreased expression of E-cadherin, keratin 6 and vimentin might be included in the mechanisms of cell migration acceleration in the absence of epiplakin. The mechanism of cell proliferation stimulation by epiplakin knockdown is to be investigated.

  20. Silencing of Prrx2 Inhibits the Invasion and Metastasis of Breast Cancer both In Vitro and In Vivo by Reversing Epithelial-Mesenchymal Transition

    Directory of Open Access Journals (Sweden)

    Zhi-Dong Lv

    2017-07-01

    Full Text Available Background/Aims: Epithelial-mesenchymal transition (EMT is recognized as a crucial mechanism in breast cancer progression and metastasis. Paired-related homeobox 2 (Prrx2 has been identified as a new EMT inducer in cancer, but the underlying mechanisms are still poorly understood. Methods: The expression of Prrx2 was assessed by immunohistochemistry in breast cancer tissues to evaluate the clinicopathological significance of Prrx2, as well as the correlation between Prrx2 and EMT. Short hairpin RNA knockdown of Prrx2 was used to examine cellular effects of Prrx2, detecte the expression of Wnt/β-catenin signaling and EMT-associated proteins, and observe cell proliferation, invasion and migration abilities in vitro and in vivo. Results: Clinical association studies showed that Prrx2 expression was related to tumor size, lymph node metastasis, tumor node metastasis stages, EMT and poor survival. Results also showed that knockdown of Prrx2 could alter cell morphology, suppressed the abilities of cell proliferation, invasion and migration in breast cancer. Moreover, silencing of Prrx2 induced the mesenchymal-epithelial transition and prevented nuclear translocation of β-catenin, inhibited wnt/β-catenin signaling pathway. Conclusion: Our study indicated that Prrx2 may be an important activator of EMT in human breast cancer and it can serve as a molecular target of therapeutic interventions for breast cancer.

  1. Taxonomy of breast cancer based on normal cell phenotype predicts outcome

    Science.gov (United States)

    Santagata, Sandro; Thakkar, Ankita; Ergonul, Ayse; Wang, Bin; Woo, Terri; Hu, Rong; Harrell, J. Chuck; McNamara, George; Schwede, Matthew; Culhane, Aedin C.; Kindelberger, David; Rodig, Scott; Richardson, Andrea; Schnitt, Stuart J.; Tamimi, Rulla M.; Ince, Tan A.

    2014-01-01

    Accurate classification is essential for understanding the pathophysiology of a disease and can inform therapeutic choices. For hematopoietic malignancies, a classification scheme based on the phenotypic similarity between tumor cells and normal cells has been successfully used to define tumor subtypes; however, use of normal cell types as a reference by which to classify solid tumors has not been widely emulated, in part due to more limited understanding of epithelial cell differentiation compared with hematopoiesis. To provide a better definition of the subtypes of epithelial cells comprising the breast epithelium, we performed a systematic analysis of a large set of breast epithelial markers in more than 15,000 normal breast cells, which identified 11 differentiation states for normal luminal cells. We then applied information from this analysis to classify human breast tumors based on normal cell types into 4 major subtypes, HR0–HR3, which were differentiated by vitamin D, androgen, and estrogen hormone receptor (HR) expression. Examination of 3,157 human breast tumors revealed that these HR subtypes were distinct from the current classification scheme, which is based on estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Patient outcomes were best when tumors expressed all 3 hormone receptors (subtype HR3) and worst when they expressed none of the receptors (subtype HR0). Together, these data provide an ontological classification scheme associated with patient survival differences and provides actionable insights for treating breast tumors. PMID:24463450

  2. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... However, troglitazone had protective effects of EPCR on injured cells. Key words: Endothelial protein C receptor, renal tubular epithelial cell, troglitazone, tumor necrosis factor-α, interleukin-1β; high glucose. ..... induce apoptosis of vascular smooth muscle cells through an extracellular signal- regulated ...

  3. Toxic mechanisms of copper oxide nanoparticles in epithelial kidney cells

    DEFF Research Database (Denmark)

    Thit, Amalie; Selck, Henriette; Bjerregaard, Henning F.

    2015-01-01

    CuO NPs have previously been reported as toxic to a range of cell cultures including kidney epithelial cells from the frog, Xenopus laevis (A6). Here we examine the molecular mechanisms affecting toxicity of Cu in different forms and particle sizes. A6 cells were exposed to ionic Cu (Cu2+) or CuO...

  4. Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate

    Directory of Open Access Journals (Sweden)

    Zvelebil Marketa

    2008-12-01

    distinct gene expression patterns. These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate. These data will form the basis for understanding not only cell fate determination and cellular homeostasis in the normal mammary epithelium but also the contribution of different mammary epithelial cell types to the etiology and molecular pathology of breast disease.

  5. Breast cancer circulating tumor cells

    Directory of Open Access Journals (Sweden)

    Maria Joao Carvalho

    2011-12-01

    Full Text Available Metastasization of breast cancer involves various mechanisms responsible for progression from invasive lesion to dissemination in distant organs. Regional lymph node metastasization was considered an initial step in this process, but it is now recognized that hematogenous dissemination is a deviation from lymphatic circulation. The detection of circulating tumor cells (CTC is an aim in several oncology areas. For this purpose, several techniques have been used to detect CTC, including the use of antibodies and techniques with nucleic acids. This study reviews the published studies considering the detection of breast cancer CTC. There are focused the difficulties in identifying a CTC in a heterogeneous population, the handling of the sample, criteria of positivity, analytical techniques, and specific markers. There are systematized various specific markers of breast cancer cells also the problems with false positive results. Finally, we hypothesize clinical applications either as a prognostic marker or as a therapeutic response monitor.

  6. Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology.

    Science.gov (United States)

    Lasalvia, Maria; Castellani, Stefano; D'Antonio, Palma; Perna, Giuseppe; Carbone, Annalucia; Colia, Anna Laura; Maffione, Angela Bruna; Capozzi, Vito; Conese, Massimo

    2016-10-15

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  8. Hair keratin KRT81 is expressed in normal and breast cancer cells and contributes to their invasiveness.

    Science.gov (United States)

    Nanashima, Naoki; Horie, Kayo; Yamada, Toshiyuki; Shimizu, Takeshi; Tsuchida, Shigeki

    2017-05-01

    Keratins are fibrous proteins. Hair keratins constitute hard structures such as the hair and nails, and cytokeratins have been used as markers of breast carcinoma. However, the expression and function of full-size hair keratin genes have not been previously demonstrated in breast cancer. We investigated the expression of the hair keratin, KRT81, and its function in human breast cancer and normal mammary epithelial cells. Western blotting showed full size 55-kDa KRT81 expression in the human breast cancer cell lines, MCF7, SKBR3 and MDA-MB-231, normal human mammary epithelial cells (HMEC), and non-neoplastic cells (MCF10A). Reverse transcription-polymerase chain reaction revealed that the full size KRT81, including its 5' region is expressed in breast cells. Immunohistochemical and immunofluorescence analyses showed that KRT81 was located in the cytoplasm. To investigate the function of KRT81, we knocked down KRT81 by siRNA in MCF10A cells. Microarray analysis revealed that the expression of genes related to invasion such as matrix metallopeptidase (MMP)9 was decreased. In KRT81-knockdown MDA-MB231 cells, zymography revealed a decrease in MMP9 activity, while scratch and invasion assays revealed that KRT81-knockdown decreased cell migration and invasion abilities. This is the first study showing that full size KRT81 is expressed in normal breast epithelial cells and breast cancer cells. Moreover, our results indicate that KRT81 contributes to the migration and invasion of breast cancer cells.

  9. Epithelial to Mesenchymal Transition Promotes Breast Cancer Progression via a Fibronectin-dependent STAT3 Signaling Pathway*

    Science.gov (United States)

    Balanis, Nikolas; Wendt, Michael K.; Schiemann, Barbara J.; Wang, Zhenghe; Schiemann, William P.; Carlin, Cathleen R.

    2013-01-01

    We previously established that overexpression of the EGF receptor (EGFR) is sufficient to induce tumor formation by otherwise nontransformed mammary epithelial cells, and that the initiation of epithelial-mesenchymal transition (EMT) is capable of increasing the invasion and metastasis of these cells. Using this breast cancer (BC) model, we find that in addition to EGF, adhesion to fibronectin (FN) activates signal transducer and activator of transcription 3 (STAT3) through EGFR-dependent and -independent mechanisms. Importantly, EMT facilitated a signaling switch from SRC-dependent EGFR:STAT3 signaling in pre-EMT cells to EGFR-independent FN:JAK2:STAT3 signaling in their post-EMT counterparts, thereby sensitizing these cells to JAK2 inhibition. Accordingly, human metastatic BC cells that failed to activate STAT3 downstream of EGFR did display robust STAT3 activity upon adhesion to FN. Furthermore, FN enhanced outgrowth in three-dimensional organotypic cultures via a mechanism that is dependent upon β1 integrin, Janus kinase 2 (JAK2), and STAT3 but not EGFR. Collectively, our data demonstrate that matrix-initiated signaling is sufficient to drive STAT3 activation, a reaction that is facilitated by EMT during BC metastatic progression. PMID:23653350

  10. Epithelial to mesenchymal transition promotes breast cancer progression via a fibronectin-dependent STAT3 signaling pathway.

    Science.gov (United States)

    Balanis, Nikolas; Wendt, Michael K; Schiemann, Barbara J; Wang, Zhenghe; Schiemann, William P; Carlin, Cathleen R

    2013-06-21

    We previously established that overexpression of the EGF receptor (EGFR) is sufficient to induce tumor formation by otherwise nontransformed mammary epithelial cells, and that the initiation of epithelial-mesenchymal transition (EMT) is capable of increasing the invasion and metastasis of these cells. Using this breast cancer (BC) model, we find that in addition to EGF, adhesion to fibronectin (FN) activates signal transducer and activator of transcription 3 (STAT3) through EGFR-dependent and -independent mechanisms. Importantly, EMT facilitated a signaling switch from SRC-dependent EGFR:STAT3 signaling in pre-EMT cells to EGFR-independent FN:JAK2:STAT3 signaling in their post-EMT counterparts, thereby sensitizing these cells to JAK2 inhibition. Accordingly, human metastatic BC cells that failed to activate STAT3 downstream of EGFR did display robust STAT3 activity upon adhesion to FN. Furthermore, FN enhanced outgrowth in three-dimensional organotypic cultures via a mechanism that is dependent upon β1 integrin, Janus kinase 2 (JAK2), and STAT3 but not EGFR. Collectively, our data demonstrate that matrix-initiated signaling is sufficient to drive STAT3 activation, a reaction that is facilitated by EMT during BC metastatic progression.

  11. Increased epithelial-free areas in thymuses with altered EphB-mediated thymocyte-thymic epithelial cell interactions.

    Science.gov (United States)

    García-Ceca, Javier; Montero-Herradón, Sara; Alfaro, David; Zapata, Agustín G

    2017-10-01

    Epithelial-free areas, present in both thymic cortex and medulla, have been studied in WT and EphB-deficient mice that have important alterations in the development of thymic epithelium due to the lack of proper thymocyte-thymic epithelial cell interactions. In both WT and mutant thymuses, the number and size of epithelial-free areas are significantly larger in the medulla than in the cortex. The two parameters show a reverse correlation: low numbers of these areas course with large epithelial-free areas and vice versa. However, their structure and cell content are similar in mutant and WT thymuses. Cortical epithelial-free areas just contain DP thymocytes, while the medullary ones consist of SP cells, blood vessels, mesenchyme-derived ER-TR7+ cells and components of the extracellular matrix (i.e., collagen IV, fibronectin, laminin). Other components, such as desmin, αSMA, PDGFRβ and Ng2, frequently associated with blood vessel walls, also appear. Vimentin, although present in medullary epithelial-free areas, does not co-express with epithelial cells. Other markers related to epithelial-mesenchymal transitions, such as Snail, Slug or FSP1, are not expressed. These results suggest that alterations in the cell interactions between distinct thymic cell components that induce both increased proportions of apoptotic thymic epithelial cells and altered behavior of the mesenchyme associated with the medullary vasculature could explain the appearance of these areas and their differences in the cortex and medulla.

  12. SEMP1, a senescence-associated cDNA isolated from human mammary epithelial cells, is a member of an epithelial membrane protein superfamily.

    Science.gov (United States)

    Swisshelm, K; Machl, A; Planitzer, S; Robertson, R; Kubbies, M; Hosier, S

    1999-01-21

    We have cloned a human cDNA, SEMP1 (senescence-associated epithelial membrane protein 1), using differential display (DD) of mRNA. We compared mRNA expression profiles between cultured normal senescent human mammary epithelial cells (HMECs) and proliferating, early passage HMECs. From the amino acid sequence of the open reading frame (ORF) of the cDNA, we infer that the protein belongs to a family of membrane-associated, epithelial cell-specific proteins. The translation product has 91% identity to a mouse protein, claudin-1, a tight junction (TJ)-associated protein. SEMP1 mRNA is expressed in human tissues, including adult and fetal liver, pancreas, placenta, adrenals, prostate and ovary but at low or undetectable levels in a number of human breast cancer cell lines. SEMP1 is a member of a superfamily of epithelial membrane proteins (EMPs), which may have multiple potential functions, including maintenance and regulation of cell polarity and permeability, perhaps through mechanisms involving tight junctions.

  13. MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer.

    Science.gov (United States)

    Chang, Chia-Wei; Yu, Jyh-Cherng; Hsieh, Yi-Hsien; Yao, Chung-Chin; Chao, Jui-I; Chen, Po-Ming; Hsieh, Hsiao-Yen; Hsiung, Chia-Ni; Chu, Hou-Wei; Shen, Chen-Yang; Cheng, Chun-Wen

    2016-03-29

    The epithelial-to-mesenchymal (EMT) transition is a prerequisite for conferring metastatic potential during tumor progression. microRNA-30a (miR-30a) expression was significantly lower in aggressive breast cancer cell lines compared with non-invasive breast cancer and non-malignant mammary epithelial cell lines. In contrast, miR-30a overexpression reversed the mesenchymal appearance of cancer cells to result in a cobblestone-like epithelial phenotype. We identified Slug, one of the master regulators of EMT, as a target of miR-30a using in silico prediction. Reporter assays indicated that miR-30a could bind to the 3'-untranslted region of Slug mRNA. Furthermore, we linked miR-30a to increased expression of claudins, a family of tight junction transmembrane proteins. An interaction between Slug and E-box in the claudin promoter sequences was reduced upon miR-30a overexpression, further leading to reduction of filopodia formation and decreased invasiveness/metastasis capabilities of breast cancer cells. Consistently, delivery of miR-30a in xenografted mice decreased tumor invasion and migration. In patients with breast cancer, a significantly elevated risk of the miR-30alow/CLDN2low/FSCNhigh genotype was observed, linking to a phenotypic manifestation of larger tumor size, lymph node metastasis, and advanced tumor stage among patients. In conclusion, the miR-30a/Slug axis inhibits mesenchymal tumor development by interfering with metastatic cancer cell programming and may be a potential target for therapy in breast cancer.

  14. No junctional communication between epithelial cells in hydra

    DEFF Research Database (Denmark)

    de Laat, S W; Tertoolen, L G; Grimmelikhuijzen, C J

    1980-01-01

    Diffusion gradients of morphogens have been inferred as a basis for the control of morphogenesis in hydra, and morphogenetic substances have been found which, on the basis of their molecular weight (MW), should be able to pass gap junctions. There have been several reports of the presence of gap...... junctions between epithelial cells of hydra. However, until now, there has been no report published on whether these junctions enable the epithelial cells to exchange molecules of small molecular weight, as has been described in other organisms. Therefore we decided to investigate the communicative...... properties of the junctional membranes by electrophysiological methods and by intracellular-dye iontophoresis. We report here that no electrotonic coupling is detectable between epithelial cells of Hydra attenuata in: (1) intact animals, (2) head-regenerating animals, (3) cell re-aggregates, and (4) hydra...

  15. High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells.

    Science.gov (United States)

    Chen, Yu-Ching; Statt, Sarah; Wu, Reen; Chang, Hao-Teng; Liao, Jiunn-Wang; Wang, Chien-Neng; Shyu, Woei-Cherng; Lee, Chen-Chen

    2016-01-07

    Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway.

  16. Identification of cell-surface markers for detecting breast cancer cells in ovarian tissue.

    Science.gov (United States)

    Peters, Inge T A; Hilders, Carina G J M; Sier, Cornelis F M; Vahrmeijer, Alexander L; Smit, Vincent T H B M; Baptist Trimbos, J; Kuppen, Peter J K

    2016-08-01

    The safety of ovarian tissue autotransplantation in oncology patients cannot be ensured, as current tumor-detection methods compromise the ovarian tissue viability. Although non-destructive methods (for instance near-infrared fluorescence imaging) can discriminate malignant from healthy tissues while leaving the examined tissues unaffected, they require specific cell-surface tumor markers. We determined which tumor markers are suitable targets for tumor-specific imaging to exclude the presence of breast cancer cells in ovarian tissue. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded specimens of ten ovaries from premenopausal patients. Additionally, we screened a tissue microarray containing tumor tissue cores from 24 breast cancer patients being eligible for ovarian tissue cryopreservation. The following cell-surface tumor markers were tested: E-cadherin, EMA (epithelial membrane antigen), Her2/neu (human epidermal growth factor receptor type 2), αvβ6 integrin, EpCAM (epithelial cell adhesion molecule), CEA (carcinoembryonic antigen), FR-α (folate receptor-alpha), and uPAR (urokinase-type plasminogen activator receptor). For each tumor, the percentage of positive breast tumor cells was measured. None of the ten ovaries were positive for any of the markers tested. However, all markers (except CEA and uPAR) were present on epithelial cells of inclusion cysts. E-cadherin was present in the majority of breast tumors: ≥90 % of tumor cells were positive for E-cadherin in 17 out of 24 tumors, and 100 % of tumor cells were positive in 5 out of 24 tumors. Of the markers tested, E-cadherin is the most suitable marker for a tumor-specific probe in ovarian tissue. Methods are required to distinguish inclusion cysts from breast tumor cells.

  17. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    Science.gov (United States)

    2012-04-01

    escape senescence and acquire genomic changes. Nature 2001;409:633–7. 10. Olsen CL, Gardie B, Yaswen P, Stampfer MR. Raf-1- induced growth arrest in...p16INK4a. Cell 88:593–602. 10. Olsen CL, Gardie B, Yaswen P, Stampfer MR (2002) Raf-1-induced growth arrest in human mammary epithelial cells is p16...Cycle 3, 244–246. Olsen CL, Gardie B, Yaswen P, Stampfer MR (2002) Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and

  18. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  19. Vitamin D Modulation of TRAIL Expression in Human Milk and Mammary Epithelial Cells.

    Science.gov (United States)

    Sambandam, Yuvaraj; Reddy, Sakamuri V; Mulligan, Jennifer L; Voelkel-Johnson, Christina; Wagner, Carol L

    2017-06-28

    The vitamin D levels in mothers affect the health status of both the mother and breastfeeding infant. Vitamin D deficient mothers' infants are prone to rickets. While tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been implicated in cellular growth/apoptosis, immune cell function and bone-resorbing osteoclast formation, the expression of TRAIL in human milk as a function of vitamin D status in mothers remains unknown. We hypothesized that vitamin D deficiency alters TRAIL protein levels in human breast milk and mammary epithelial cells. Milk from vitamin D deficient mothers showed high levels of TRAIL (α and β) proteins compared to milk from vitamin D replete women. Western blot analysis of total cell lysate obtained from normal human mammary epithelial (HME-1) cells treated with variable doses (0-20 nM) of vitamin D for 24 h demonstrated that low levels (0.5 to 5 nM) significantly increased the TRAIL α but no change in β expression. In contrast, vitamin D at 20 nM concentration suppressed the expression of both TRAIL α and β proteins. Consistently, vitamin D regulated TRAIL mRNA expression in HME-1 cells. Our results indicate that vitamin D status in mothers modulates TRAIL expression in breast milk, which may have implications for both mother and infant health.

  20. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

    Science.gov (United States)

    Hosoki, Koa; Kainuma, Keigo; Toda, Masaaki; Harada, Etsuko; Chelakkot-Govindalayathila, Ayshwarya-Lakshmi; Roeen, Ziaurahman; Nagao, Mizuho; D'Alessandro-Gabazza, Corina N; Fujisawa, Takao; Gabazza, Esteban C

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Cyclooxygenase-2 is a target gene of rho GDP dissociation inhibitor beta in breast cancer cells.

    NARCIS (Netherlands)

    Schunke, D.; Span, P.N.; Ronneburg, H.; Dittmer, A.; Vetter, M.; Holzhausen, H.J.; Kantelhardt, E.; Krenkel, S.; Muller, V.; Sweep, C.G.J.; Thomssen, C.; Dittmer, J.

    2007-01-01

    Rho GDP dissociation inhibitor beta (Rho-GDI beta), an inhibitor of Rho GTPases, is primarily expressed by hematopoietic cells but is also found in epithelial cancer cells. Recently, we have identified Rho-GDI beta as a target of the transcription factor Ets1. Here, we show that, in breast cancer

  2. Repression of mammosphere formation in breast cancer cells by soy isoflavone genistein and blueberry polyphenols

    Science.gov (United States)

    Epidemiological evidence implicates diets rich in fruits and vegetables in breast cancer prevention due to their phytochemical components, yet mechanisms for their anti-tumor activities are not well-understood. A small population of mammary epithelial cells, termed cancer stem cells (CSC), may be re...

  3. The origin of epithelial neoplasms after allogeneic stem cell transplantation.

    NARCIS (Netherlands)

    Smith, M.J.; Cleef, P.H. van; Schattenberg, A.V.M.B.; Krieken, J.H.J.M. van

    2006-01-01

    We analyzed five women, who have developed epithelial neoplasms after sex-mismatched stem cell transplants. Using in situ hybridization for sex chromosome-specific DNA probes and immunohistochemistry we identified the origin of the tumor cells. We conclude that none of the non-hematologic

  4. Effect of Helicobacter pylori on gastric epithelial cells

    Science.gov (United States)

    Alzahrani, Shatha; Lina, Taslima T; Gonzalez, Jazmin; Pinchuk, Irina V; Beswick, Ellen J; Reyes, Victor E

    2014-01-01

    The gastrointestinal epithelium has cells with features that make them a powerful line of defense in innate mucosal immunity. Features that allow gastrointestinal epithelial cells to contribute in innate defense include cell barrier integrity, cell turnover, autophagy, and innate immune responses. Helicobacter pylori (H. pylori) is a spiral shape gram negative bacterium that selectively colonizes the gastric epithelium of more than half of the world’s population. The infection invariably becomes persistent due to highly specialized mechanisms that facilitate H. pylori’s avoidance of this initial line of host defense as well as adaptive immune mechanisms. The host response is thus unsuccessful in clearing the infection and as a result becomes established as a persistent infection promoting chronic inflammation. In some individuals the associated inflammation contributes to ulcerogenesis or neoplasia. H. pylori has an array of different strategies to interact intimately with epithelial cells and manipulate their cellular processes and functions. Among the multiple aspects that H. pylori affects in gastric epithelial cells are their distribution of epithelial junctions, DNA damage, apoptosis, proliferation, stimulation of cytokine production, and cell transformation. Some of these processes are initiated as a result of the activation of signaling mechanisms activated on binding of H. pylori to cell surface receptors or via soluble virulence factors that gain access to the epithelium. The multiple responses by the epithelium to the infection contribute to pathogenesis associated with H. pylori. PMID:25278677

  5. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    The endothelial protein C receptor (EPCR) plays an important role within the protein C pathway in regulating coagulation and inflammation. It was reported that EPCR was expressed in large vessels, placenta, heart, liver and lung endothelial cell. However, there are a few studies concerned about renal epithelial cells.

  6. Interaction of E-cadherin and PTEN regulates morphogenesis and growth arrest in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Fournier, Marcia V.; Fata, Jimmie E.; Martin, Katherine J.; Yaswen, Paul; Bissell, Mina J.

    2009-06-03

    PTEN is a dual function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two non-malignant human mammary epithelial cell lines (HMECs) that form polarized, growth-arrested structures (acini) when cultured in 3-dimensional laminin-rich extracellular matrix gels (3D lrECM). As acini begin to form, PTEN accumulates in both the cytoplasm, and at cell-cell contacts where it colocalizes with E-cadherin/{beta}-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in SKBR3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in 3D lrECM indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus appears to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

  7. Hornerin, an S100 family protein, is functional in breast cells and aberrantly expressed in breast cancer

    Directory of Open Access Journals (Sweden)

    Fleming Jodie M

    2012-06-01

    Full Text Available Abstract Background Recent evidence suggests an emerging role for S100 protein in breast cancer and tumor progression. These ubiquitous proteins are involved in numerous normal and pathological cell functions including inflammatory and immune responses, Ca2+ homeostasis, the dynamics of cytoskeleton constituents, as well as cell proliferation, differentiation, and death. Our previous proteomic analysis demonstrated the presence of hornerin, an S100 family member, in breast tissue and extracellular matrix. Hornerin has been reported in healthy skin as well as psoriatic and regenerating skin after wound healing, suggesting a role in inflammatory/immune response or proliferation. In the present study we investigated hornerin’s potential role in normal breast cells and breast cancer. Methods The expression levels and localization of hornerin in human breast tissue, breast tumor biopsies, primary breast cells and breast cancer cell lines, as well as murine mammary tissue were measured via immunohistochemistry, western blot analysis and PCR. Antibodies were developed against the N- and C-terminus of the protein for detection of proteolytic fragments and their specific subcellular localization via fluorescent immunocytochemisty. Lastly, cells were treated with H2O2 to detect changes in hornerin expression during induction of apoptosis/necrosis. Results Breast epithelial cells and stromal fibroblasts and macrophages express hornerin and show unique regulation of expression during distinct phases of mammary development. Furthermore, hornerin expression is decreased in invasive ductal carcinomas compared to invasive lobular carcinomas and less aggressive breast carcinoma phenotypes, and cellular expression of hornerin is altered during induction of apoptosis. Finally, we demonstrate the presence of post-translational fragments that display differential subcellular localization. Conclusions Our data opens new possibilities for hornerin and its

  8. Epithelial-to-Mesenchymal Transition Contributes to Immunosuppression in Breast Carcinomas.

    Science.gov (United States)

    Dongre, Anushka; Rashidian, Mohammad; Reinhardt, Ferenc; Bagnato, Aaron; Keckesova, Zuzana; Ploegh, Hidde L; Weinberg, Robert A

    2017-08-01

    The epithelial-to-mesenchymal transition (EMT) is a cell biological program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. It is unclear, however, whether the activation of EMT in carcinoma cells can change their susceptibility to immune attack. We demonstrate here that mammary tumor cells arising from more epithelial carcinoma cell lines expressed high levels of MHC-I, low levels of PD-L1, and contained within their stroma CD8+ T cells and M1 (antitumor) macrophages. In contrast, tumors arising from more mesenchymal carcinoma cell lines exhibiting EMT markers expressed low levels of MHC-I, high levels of PD-L1, and contained within their stroma regulatory T cells, M2 (protumor) macrophages, and exhausted CD8+ T cells. Moreover, the more mesenchymal carcinoma cells within a tumor retained the ability to protect their more epithelial counterparts from immune attack. Finally, epithelial tumors were more susceptible to elimination by immunotherapy than corresponding mesenchymal tumors. Our results identify immune cells and immunomodulatory markers that can be potentially targeted to enhance the susceptibility of immunosuppressive tumors to various therapeutic regimens. Cancer Res; 77(15); 3982-9. ©2017 AACR. ©2017 American Association for Cancer Research.

  9. Glutathione Transferase as a Potential Marker for Gut Epithelial Injury versus the Protective Role of Breast Milk sIgA in Infants with Rota Virus Gastroenteritis

    Directory of Open Access Journals (Sweden)

    Lobna S. Sherif

    2015-11-01

    CONCLUSION: Breast feeding should be encouraged and highly recommended in the first two years of life as it provides Secretory IgA to breast fed infants who in turn protect them against epithelial damage caused by Rota viral gastroenteritis.

  10. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

    OpenAIRE

    Sanaz Gidfar; Farnoud Y. Milani; Milani, Behrad Y.; Xiang Shen; Medi Eslani; Ilham Putra; Michael J. Huvard; Hossein Sagha; Djalilian, Ali R.

    2017-01-01

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated ?-Galactosid...

  11. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    Science.gov (United States)

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  12. Factors influencing the exfoliation of cervicovaginal epithelial cells.

    Science.gov (United States)

    Basu, J; Mikhail, M S; Palan, P R; Payraudeau, P H; Romney, S L

    1992-12-01

    Our objective was to investigate the association of smoking and exfoliation of cervicovaginal epithelial cells while controlling for other factors that may potentially influence cell exfoliation (e.g., presence of cervical intraepithelial neoplasia or koilocytes, the use of oral contraceptives, age, and the phase of the menstrual cycle). Cervicovaginal lavage specimens and epidemiologic questionnaires were obtained with informed consent from 190 women. The cervicovaginal lavage samples were processed to separate other contaminants. The number of squamous epithelial cells counted was expressed as cells per milliliter of lavage. Multiple linear regression analysis revealed that the number of exfoliated epithelial cells was significantly higher in smokers (p < 0.01) and also in women with cervical intraepithelial neoplasia (p < 0.05). The other studied variables had no detectable effect. The findings suggest that smoking or the presence of cervical intraepithelial neoplasia may induce an acceleration in the exfoliation of cervicovaginal epithelial cells. This may alter cell maturation and may be a factor in the oncogenic process.

  13. ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC.

    Science.gov (United States)

    Griffin, Nicolas I; Sharma, Gayatri; Zhao, Xiangshan; Mirza, Sameer; Srivastava, Shashank; Dave, Bhavana J; Aleskandarany, Mohammed; Rakha, Emad; Mohibi, Shakur; Band, Hamid; Band, Vimla

    2016-11-16

    We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized. We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients. Overexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 and c-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients' outcome was independent of tumor grade

  14. The effect of TGF-beta-induced epithelial-mesenchymal transition on the expression of intracellular calcium-handling proteins in T47D and MCF-7 human breast cancer cells.

    Science.gov (United States)

    Mahdi, Shah H A; Cheng, Huanyi; Li, Jinfeng; Feng, Renqing

    2015-10-01

    The contribution of Ca(2+) in TGF-β-induced EMT is poorly understood. We aimed to confirm the effect of TGF-β on the gene expression of intracellular calcium-handling proteins and to investigate the potential underlying mechanisms in TGF-β-induced EMT. T47D and MCF-7 cells were cultured in vitro and treated with TGF-β. The mRNA expression of EMT marker genes and intracellular calcium-handling proteins were quantified by qRT-PCR. qRT-PCR and Western blot analysis results verified the changes of EMT marker gene expression. Furthermore, we found that TGF-β induced cell morphological changes significantly with an increase of cell surface area and cell length. These results indicated that TGF-β induced EMT. The mRNA expression levels of SPCA1, SPCA2 and MCU were not influenced by TGF-β treatment, while NCX1 expression was decreased in T47D cells. In addition, the mRNA levels of SERCAs and IP3Rs were significantly changed due to TGF-β-induced EMT. The TGF-β-treated T47D cells exhibited markedly greater response to ATP than the control cells, and the descent velocity of cytosolic calcium concentration was faster in TGF-β-treated cells than in control cells. This is the first report to demonstrate that TGF-β-induced EMT in human breast cancer cells is associated with alterations in endoplasmic reticulum calcium homeostasis. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

    Energy Technology Data Exchange (ETDEWEB)

    Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.; Shyamala, G.; Moses, Harold L.; Barcellos-Hoff, Mary Helen

    2005-03-03

    Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha} co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.

  16. Osmosignaling and volume regulation in intestinal epithelial cells.

    Science.gov (United States)

    Lim, Christina H; Bot, Alice G M; de Jonge, Hugo R; Tilly, Ben C

    2007-01-01

    Most cells have to perform their physiological functions under a variable osmotic stress, which, because of the relatively high permeability of the plasma membrane for water, may result in frequent alterations in cell size. Intestinal epithelial cells are especially prone to changes in cell volume because of their high capacity of salt and water transport and the high membrane expression of various nutrient transporters. Therefore, to avoid excessive shrinkage or swelling, enterocytes, like most cell types, have developed efficient mechanisms to maintain osmotic balance. This chapter reviews selected model systems that can be used to investigate cell volume regulation in intestinal epithelial cells, with emphasis on the regulatory volume decrease, and the methods available to study the compensatory redistribution of (organic) osmolytes. In addition, a brief summary is presented of the pathways involved in osmosensing and osmosignaling in the intestine.

  17. Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells

    NARCIS (Netherlands)

    Kistemaker, Loes E. M.; Hiemstra, Pieter S.; Bos, I. Sophie T.; Bouwman, Susanne; van den Berge, Maarten; Hylkema, Machteld N.; Meurs, Herman; Kerstjens, Huib A. M.; Gosens, Reinoud

    BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct

  18. Nicotine transport in lung and non-lung epithelial cells.

    Science.gov (United States)

    Takano, Mikihisa; Kamei, Hidetaka; Nagahiro, Machi; Kawami, Masashi; Yumoto, Ryoko

    2017-11-01

    Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Characteristics of [3H]nicotine uptake was studied using these cell lines. Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Of mice and women: a comparative tissue biology perspective of breast stem cells and differentiation.

    Science.gov (United States)

    Dontu, Gabriela; Ince, Tan A

    2015-06-01

    Tissue based research requires a background in human and veterinary pathology, developmental biology, anatomy, as well as molecular and cellular biology. This type of comparative tissue biology (CTB) expertise is necessary to tackle some of the conceptual challenges in human breast stem cell research. It is our opinion that the scarcity of CTB expertise contributed to some erroneous interpretations in tissue based research, some of which are reviewed here in the context of breast stem cells. In this article we examine the dissimilarities between mouse and human mammary tissue and suggest how these may impact stem cell studies. In addition, we consider the differences between breast ducts vs. lobules and clarify how these affect the interpretation of results in stem cell research. Lastly, we introduce a new elaboration of normal epithelial cell types in human breast and discuss how this provides a clinically useful basis for breast cancer classification.

  20. Inhibitory effects of ameloblastin on epithelial cell proliferation.

    Science.gov (United States)

    Saito, Noriko; Ariyoshi, Wataru; Okinaga, Toshinori; Kamegawa, Mariko; Matsukizono, Miho; Akebiyama, Yasuo; Kitamura, Chiaki; Nishihara, Tatsuji

    2014-08-01

    Ameloblastin is an enamel matrix protein expressed in several tissues. Many potential mechanisms have been identified by which ameloblastin functions as an extracellular matrix protein. However, the biological effects of ameloblastin on gingival epithelial cells remain unclear. In the present study, we established a novel system to purify recombinant human ameloblastin and clarified its biological functions in epithelial cells in vitro. Recombinant human ameloblastin was isolated from COS-7 cells overexpressing HaloTag-fused human ameloblastin by the HaloTag system and then purified further by reverse-phase high-performance liquid chromatography. SCC-25 cells, derived from human oral squamous cell carcinoma, were treated with recombinant ameloblastin and then cell survival was assessed by a WST-1 assay. Cell cycle analysis was performed by flow cytometry. The novel purification system allowed effective recovery of the recombinant ameloblastin proteins at a high purity. Recombinant ameloblastin protein was found to suppress the proliferation of SCC-25 cells. Flow cytometric analysis showed that ameloblastin treatment induced cell cycle arrest G1 phase. We developed a procedure for production of highly purified recombinant human ameloblastin. Biological analyses suggest that ameloblastin induces cell cycle arrest in epithelial cells and regulates the progression of periodontitis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Soo Hwa [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of); Choi, Changsun [Department of Food and Nutrition, College of Human Ecology, Chung-Ang University, Anseong, Gyeonggi (Korea, Republic of); Hong, Seong-Geun; Yarishkin, Oleg V. [Department of Physiology, College of Medicine, Gyeongsang National University, Jinju (Korea, Republic of); Bae, Young Min; Kim, Jae Gon [Department of Physiology, College of Medicine, Konkuk University, Seoul (Korea, Republic of); O' Grady, Scott M. [Department of Physiology, 495 Animal Science/Veterinary Medicine Bldg., St. Paul, University of Minnesota, MN (United States); Yoon, Kyong-Ah [Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi (Korea, Republic of); Kang, Kyung-Sun [Veterinary Public Health, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul (Korea, Republic of); Ryu, Pan Dong [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of); Lee, So Yeong, E-mail: leeso@snu.ac.kr [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of)

    2009-06-26

    Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.

  2. Oxidative Stress, Cell Death, and Other Damage to Alveolar Epithelial Cells Induced by Cigarette Smoke

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    Nagai A

    2003-09-01

    Full Text Available Abstract Cigarette smoking is a major risk factor in the development of various lung diseases, including pulmonary emphysema, pulmonary fibrosis, and lung cancer. The mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following cigarette smoking, alterations in alveolar epithelial cells induce an increase in epithelial permeability, a decrease in surfactant production, the inappropriate production of inflammatory cytokines and growth factors, and an increased risk of lung cancer. However, the most deleterious effect of cigarette smoke on alveolar epithelial cells is cell death, i.e., either apoptosis or necrosis depending on the magnitude of cigarette smoke exposure. Cell death induced by cigarette smoke exposure can largely be accounted for by an enhancement in oxidative stress. In fact, cigarette smoke contains and generates many reactive oxygen species that damage alveolar epithelial cells. Whether apoptosis and/or necrosis in alveolar epithelial cells is enhanced in healthy cigarette smokers is presently unclear. However, recent evidence indicates that the apoptosis of alveolar epithelial cells and alveolar endothelial cells is involved in the pathogenesis of pulmonary emphysema, an important cigarette smoke-induced lung disease characterized by the loss of alveolar structures. This review will discuss oxidative stress, cell death, and other damage to alveolar epithelial cells induced by cigarette smoke.

  3. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    Science.gov (United States)

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-05-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices. Failure of most biomaterials originates from the inability to predict and control the influence of their surface properties on biological phenomena, particularly protein adsorption, and cellular behaviour, which subsequently results in unfavourable host response. Here, we introduce a surface-proteomic screening approach using a label-free mass spectrometry technique to decipher the adsorption profile of extracellular matrix (ECM) proteins on different biomaterials, and correlate it with cellular behaviour. We demonstrated that the way a biomaterial selectively interacts with specific ECM proteins of a given tissue seems to determine the interactions between the cells of that tissue and biomaterials. Accordingly, this approach can

  4. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... block alloactivation, had no inhibitory effect on RPE-mediated T-cell apoptotic responses in MHC class II-specific CD4+ T-cell lines. CONCLUSIONS: Retinal pigment epithelial cells express FasL and induce TCR-independent apoptosis in activated human T cells through Fas-FasL interaction. Retinal pigment...

  5. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    block alloactivation, had no inhibitory effect on RPE-mediated T-cell apoptotic responses in MHC class II-specific CD4+ T-cell lines. CONCLUSIONS: Retinal pigment epithelial cells express FasL and induce TCR-independent apoptosis in activated human T cells through Fas-FasL interaction. Retinal pigment...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  6. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.

    Science.gov (United States)

    Padilla-Nash, Hesed M; McNeil, Nicole E; Yi, Ming; Nguyen, Quang-Tri; Hu, Yue; Wangsa, Danny; Mack, David L; Hummon, Amanda B; Case, Chanelle; Cardin, Eric; Stephens, Robert; Difilippantonio, Michael J; Ried, Thomas

    2013-08-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.

  7. Apicobasal Polarity Controls Lymphocyte Adhesion to Hepatic Epithelial Cells

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    Natalia Reglero-Real

    2014-09-01

    Full Text Available Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1 adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α. We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

  8. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    by inducible nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells. METHODS: A colonic cell line (HT29) was stimulated for 1-10 weeks with interferon...... stimulated cells had increased DNA instability (Pcytokine exposure induces an iNOS dependent up-regulation of ROS production and DNA instability. This mechanism...

  9. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    Science.gov (United States)

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-03-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  10. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Science.gov (United States)

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel. PMID:23538640

  11. Myc mediates cancer stem-like cells and EMT changes in triple negative breast cancers cells.

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    Shuping Yin

    Full Text Available Women with triple negative breast cancer (TNBC have poor prognosis compared to other breast cancer subtypes. There were several reports indicating racial disparity in breast cancer outcomes between African American (AA and European American (EA women. For example, the mortality rates of AA breast cancer patients were three times higher than of EA patients, even though, the incidence is lower in AA women. Our in vitro studies indicate that cancer stem-like cells (CSCs derived from AA TNBC cell lines have significantly higher self-renewal potential (mammosphere formation than CSCs derived from EA cell lines. TNBC tumors express high levels of Myc compared to luminal A or HER2 expressing breast cancers. We studied the effects of c-Myc overexpression on CSCs and chemotherapy in AA, and EA derived TNBC cell line(s. Overexpression of c-Myc in AA derived MDA-MB-468 (Myc/MDA-468 cells resulted in a significant increase in CSCs and with minimal changes in epithelial-to-mesenchymal transition (EMT compared to the control group. In contrast, overexpression of c-Myc in EA derived MDA-MB-231(Myc/MDA-231 cells led to increased epithelial-to-mesenchymal transition (EMT, with a minimal increase in CSCs compared to the control group. Myc/MDA-468 cells were resistant to standard chemotherapeutic treatments such as iniparib (PARP inhibitor plus cisplatin, / iniparib, cisplatin, paclitaxel and docetaxel. However, Myc/MDA-231 cells, which showed EMT changes responded to iniparib with cisplatin, but were resistant to other drugs, such as iniparib, cisplatin, paclitaxel and docetaxel. Collectively, our results indicate that intrinsic differences in the tumor biology may contribute to the breast cancer disparities.

  12. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    Science.gov (United States)

    Spivey, Megan A; Dunn-Horrocks, Sadie L; Duong, Tri

    2014-11-01

    Administration of probiotic Lactobacillus cultures is an important alternative to the use of antibiotic growth promoters and has been demonstrated to improve animal health, growth performance, and preharvest food safety in poultry production. Whereas gastrointestinal colonization is thought to be critical to their probiotic functionality, factors important to Lactobacillus colonization in chickens are not well understood. In this study we investigate epithelial cell adhesion in vitro and colonization of Lactobacillusin vivo in broiler chickens. Adhesion of Lactobacillus cultures to epithelial cells was evaluated using the chicken LMH cell line. Lactobacillus cultures were able adhere effectively to LMH cells relative to Bacillus subtilis and Salmonella Typhimurium. Epithelial cell adhesion was similar for Lactobacillus crispatus TDCC 75, L. cristpatus TDCC 76, and Lactobacillus gallinarum TDCC 77, and all 3 were more adherent than L. gallinarum TDCC 78. However, when colonization was evaluated in the ileum and cecum of broiler chicks, L. crispatus TDCC 75 and L. gallinarum TDCC 77 were more persistent than L. crispatus TDCC 76 and L. gallinarum TDCC 78. The reduction of growth in medium supplemented with oxgal was greater for L. gallinarum TDCC 78 than L. gallinarum TDCC 77, suggesting that whereas adhesion was similar for the 2 strains, the difference in colonization between L. gallinarum strains may be due in part to their bile sensitivity. This study demonstrates that whereas adhesion to epithelial cells may be important in predicting gastrointestinal colonization, other factors including bile tolerance may also contribute to the colonization of Lactobacillus in poultry. Additionally, the chicken LMH cell line is expected to provide a platform for investigating mechanisms of Lactobacillus adhesion to epithelial tissue and evaluating the probiotic potential Lactobacillus in poultry. ©2014 Poultry Science Association Inc.

  13. Renal epithelial cells can release ATP by vesicular fusion

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    Randi G Bjaelde

    2013-09-01

    Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

  14. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

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    Tomoyo Yoshinaga

    Full Text Available Epithelial-mesenchymal transition (EMT of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1 and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1 and an agonist for the G protein-coupled receptor 55 (GRP55, the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

  15. Sodium selectivity of semicircular canal duct epithelial cells

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    Harbidge Donald G

    2011-09-01

    Full Text Available Abstract Background Sodium absorption by semicircular canal duct (SCCD epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC, comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197, whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. Conclusions These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the

  16. Cancer Stem Cells, Epithelial to Mesenchymal Markers, and Circulating Tumor Cells in Small Cell Lung Cancer

    NARCIS (Netherlands)

    Pore, Milind; Meijer, Coby; de Bock, Geertruida H.; Boersma-van Ek, Wytske; Terstappen, Leon W. M. M.; Groen, Harry J. M.; Timens, Wim; Kruyt, Frank A. E.; Hiltermann, T. Jeroen N.

    2016-01-01

    The prognostic value of markers of cancer stem cells and epithelial to mesenchymal transition in small cell lung cancer is not known. We retrospectively studied these markers in the biopsy tissue of patients with small cell lung cancer and correlated them with overall survival and the strongest

  17. Differential expression of WISP-1 and WISP-2 genes in normal and transformed human breast cell lines.

    Science.gov (United States)

    Saxena, N; Banerjee, S; Sengupta, K; Zoubine, M N; Banerjee, S K

    2001-12-01

    The transcriptional alterations of specific gene(s) are actively associated with the development of different cancers including breast. The preceding studies of different laboratories documented at least 40 genes that may contribute directly to the genesis of cancer. Using differential display, RT-PCR and DNA sequencing analyses in normal human mammary epithelial cells (HMEC) and various breast tumor cell lines including MCF-7, ZR-75, T-47D and SKBR2, we demonstrated that WISP-1 and WISP-2 genes are differentially transcribed in these cells. WISP-2 mRNA transcription was identified in all 4 tumor derived cell lines, but the mRNA expression was undetected or minimally detected in normal breast epithelial cells. WISP-1 mRNA expression was identified in normal and transformed cell lines. However, the level of expression was higher in different breast tumor cell lines as compared to HMEC. The mRNA expression profiles of WISP genes in normal breast epithelial cells and breast tumor derived cell lines indicated a strong possibility of the involvement of WISP-signaling in the development of human breast tumors, and can be utilized as genetic markers of this disease.

  18. [STR genotyping from trace epithelial cells on fountain pen].

    Science.gov (United States)

    Yang, Fan; Mei, Shan-Zong; Li, Yong-Hong; Feng, Yan; Yu, Wei-Dong; Zhang, Yue

    2008-02-01

    To evaluate the feasibility of STR genotyping from trace epithelial cells on fountain pen and to discuss the impact of conservation time on DNA typing. Seven fountain pens were separately used by each of the 17 volunteers 20 minutes per day for a month and then were preserved on day 1, 3, 5, 7, 14, 21, and 28. DNA was extracted from the epithelial cells on fountain pen by silicon bead and was genotyped by Identifier kit. The corresponding control samples were buccal swabs of the above volunteers. The detectable numbers of loci were counted for assessment. There were statistically significant differences in the DNA genotyping by detectable numbers of gene loci between buccal swabs and epithelial cells on fountain pen of different conservation times (P fountain pen preserved on day 1, 3, 5, 7, 14, 21, 28 and the corresponding oral swabs were also statistically significant (P fountain pen if the tests were performed within 24 hours. The trace epithelial cells on fountain pen can be used as biological samples for personal identification, but the conservation time would have influence on the results of DNA genotyping.

  19. Membrane dynamics and the regulation of epithelial cell polarity

    NARCIS (Netherlands)

    van der Wouden, JM; Maier, O; van IJzendoorn, SCD; Hoekstra, D

    2003-01-01

    Plasma membranes of epithelial cells consist of two domains, an apical and a basolateral domain, the surfaces of which differ in composition. The separation of these domains by a tight junction and the fact that specific transport pathways exist for intracellular communication between these domains

  20. Computational investigation of epithelial cell dynamic phenotype in vitro

    Directory of Open Access Journals (Sweden)

    Debnath Jayanta

    2009-05-01

    Full Text Available Abstract Background When grown in three-dimensional (3D cultures, epithelial cells typically form cystic organoids that recapitulate cardinal features of in vivo epithelial structures. Characterizing essential cell actions and their roles, which constitute the system's dynamic phenotype, is critical to gaining deeper insight into the cystogenesis phenomena. Methods Starting with an earlier in silico epithelial analogue (ISEA1 that validated for several Madin-Darby canine kidney (MDCK epithelial cell culture attributes, we built a revised analogue (ISEA2 to increase overlap between analogue and cell culture traits. Both analogues used agent-based, discrete event methods. A set of axioms determined ISEA behaviors; together, they specified the analogue's operating principles. A new experimentation framework enabled tracking relative axiom use and roles during simulated cystogenesis along with establishment of the consequences of their disruption. Results ISEA2 consistently produced convex cystic structures in a simulated embedded culture. Axiom use measures provided detailed descriptions of the analogue's dynamic phenotype. Dysregulating key cell death and division axioms led to disorganized structures. Adhering to either axiom less than 80% of the time caused ISEA1 to form easily identified morphological changes. ISEA2 was more robust to identical dysregulation. Both dysregulated analogues exhibited characteristics that resembled those associated with an in vitro model of early glandular epithelial cancer. Conclusion We documented the causal chains of events, and their relative roles, responsible for simulated cystogenesis. The results stand as an early hypothesis–a theory–of how individual MDCK cell actions give rise to consistently roundish, cystic organoids.

  1. The Role of SnoN and Ski in Mammary Epithelial Cell Transformation

    National Research Council Canada - National Science Library

    Pan, Deng

    2007-01-01

    .... Higher level of Ski/SnoN is found in transformed mammary epithelial cells. Ski/SnoN might play a role in regulation of the transformation of mammary epithelial cell by antagonizing TGF signaling pathway...

  2. Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies.

    Directory of Open Access Journals (Sweden)

    Surendran Thavagnanam

    Full Text Available INTRODUCTION: Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures. METHODS: Paired nasal and bronchial epithelial cells from asthmatic children (n = 9 were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis. RESULTS: Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13. CONCLUSIONS: We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.

  3. Cellular Effect of Curcumin and Citral Combination on Breast Cancer Cells: Induction of Apoptosis and Cell Cycle Arrest.

    Science.gov (United States)

    Patel, Pinaki B; Thakkar, Vasudev R; Patel, Jagdish S

    2015-09-01

    The unmanageable side effects caused by current chemotherapy regimens to treat cancer are an unresolved problem. Although many phytonutrients are useful as chemoprevention without side effects, their effects are slower and smaller than conventional chemotherapy. In the present work, we examined the cumulative effect of two phytonutrients, curcumin and citral, on breast cancer cell lines and compared their effect with the known chemotherapy regimen of cyclophosphamide, methotrexate, and 5-fluorouracil. Using cultured breast cancer and normal epithelial cells, the cytotoxic and apoptotic effect of curcumin and citral was evaluated in vitro. The synergistic effect of curcumin and citral was calculated by a combination index study using the method by Chou and Talalay. Cell death pathways and mechanisms were analyzed by measuring intracellular reactive oxygen species (ROS) and apoptotic protein levels. Curcumin and citral caused dose and time dependent cell death and showed a synergistic effect at effective concentration EC50 and above concentrations in breast cancer cells without disturbing normal breast epithelial cells. With combination curcumin and citral treatment, apoptosis induction and cell cycle arrest at G0/G1 phase in breast cancer cells were observed. Curcumin and citral generated ROS and activated p53 and poly (ADP-ribose) polymerase-1 mediated apoptotic pathways. The results of this study suggest that curcumin and citral in combination may be a useful therapeutic intervention for breast cancer.

  4. In search of a stem cell hierarchy in the human breast and its relevance to breast cancer evolution

    DEFF Research Database (Denmark)

    Villadsen, René

    2005-01-01

    of the concept of tumors as caricatures of tissue renewal. Thus, recent molecular classification of breast cancer based on genome wide expression analysis operates with different subtypes with specific reference to the normal luminal epithelial and myoepithelial/basal lineages in the breast. Apparently some...... tumors are lineage restricted and others differentiate more broadly as if they have preserved some stem-like properties. This holds promise for the existence of a stem cell hierarchy, the understanding of which may prove to be instrumental in further dissecting the histogenesis of breast cancer evolution...... tumor progression is still very much required. Complete knowledge of the primary cell of origin of breast cancer and the mechanisms that influence differentiation programs during tumor initiation, promotion and progression may be crucial for the development of novel non-toxic therapies that influence...

  5. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    Science.gov (United States)

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation. Copyright © 2016. Published by Elsevier Inc.

  6. Interplay of Stem Cell Characteristics, EMT, and Microtentacles in Circulating Breast Tumor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Charpentier, Monica [Program in Molecular Medicine, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-20, Baltimore, MD 21201 (United States); Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-29, Baltimore, MD 21201 (United States); Martin, Stuart, E-mail: ssmartin@som.umaryland.edu [Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-29, Baltimore, MD 21201 (United States); Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-29, Baltimore, MD 21201 (United States)

    2013-11-14

    Metastasis, not the primary tumor, is responsible for the majority of breast cancer-related deaths. Emerging evidence indicates that breast cancer stem cells (CSCs) and the epithelial-to-mesenchymal transition (EMT) cooperate to produce circulating tumor cells (CTCs) that are highly competent for metastasis. CTCs with both CSC and EMT characteristics have recently been identified in the bloodstream of patients with metastatic disease. Breast CSCs have elevated tumorigenicity required for metastatic outgrowth, while EMT may promote CSC character and endows breast cancer cells with enhanced invasive and migratory potential. Both CSCs and EMT are associated with a more flexible cytoskeleton and with anoikis-resistance, which help breast carcinoma cells survive in circulation. Suspended breast carcinoma cells produce tubulin-based extensions of the plasma membrane, termed microtentacles (McTNs), which aid in reattachment. CSC and EMT-associated upregulation of intermediate filament vimentin and increased detyrosination of α-tubulin promote the formation of McTNs. The combined advantages of CSCs and EMT and their associated cytoskeletal alterations increase metastatic efficiency, but understanding the biology of these CTCs also presents new therapeutic targets to reduce metastasis.

  7. Biomechanics of epithelial cell islands analyzed by modeling and experimentation

    CERN Document Server

    Coburn, Luke; Noppe, Adrian; Caldwell, Benjamin J; Moussa, Elliott; Yap, Chloe; Priya, Rashmi; Lobaskin, Vladimir; Roberts, Anthony P; Yap, Alpha S; Neufeld, Zoltan; Gomez, Guillermo A

    2016-01-01

    We generated a new computational approach to analyze the biomechanics of epithelial cell islands that combines both vertex and contact-inhibition-of-locomotion models to include both cell-cell and cell-substrate adhesion. Examination of the distribution of cell protrusions (adhesion to the substrate) in the model predicted high order profiles of cell organization that agree with those previously seen experimentally. Cells acquired an asymmetric distribution of protrusions (and traction forces) that decreased when moving from the edge to the island center. Our in silico analysis also showed that tension on cell-cell junctions (and monolayer stress) is not homogeneous across the island. Instead it is higher at the island center and scales up with island size, which we confirmed experimentally using laser ablation assays and immunofluorescence. Moreover, our approach has the minimal elements necessary to reproduce mechanical crosstalk between both cell-cell and cell substrate adhesion systems. We found that an i...

  8. Circulating tumor cell clusters-associated gene plakoglobin and breast cancer survival.

    Science.gov (United States)

    Lu, Lingeng; Zeng, Hongmei; Gu, Xinsheng; Ma, Wenxue

    2015-06-01

    Breast cancer recurrence is a major cause of the disease-specific death. Circulating tumor cells (CTCs) are negatively associated with breast cancer survival. Plakoglobin, a cell adhesion protein, was recently reported as a determinant of CTCs types, single or clustered ones. Here, we aim to summarize the studies on the roles of plakoglobin and evaluate the association of plakoglobin and breast cancer survival. Plakoglobin as a key component in both cell adhesion and the signaling pathways was briefly reviewed first. Then the double-edge functions of plakoglobin in tumors and its association with CTCs and breast cancer metastasis were introduced. Finally, based on an open-access database, the association between plakoglobin and breast cancer survival was investigated using univariate and multivariate survival analyses. Plakoglobin may be a molecule functioning as a double-edge sword. Loss of plakoglobin expression leads to increased motility of epithelial cells, thereby promoting epithelial-mesenchymal transition and further metastasis of cancer. However, studies also show that plakoglobin can function as an oncogene. High expression of plakoglobin results in clustered tumor cells in circulation with high metastatic potential in breast cancer and shortened patient survival. Plakoglobin may be a potential prognostic biomarker that can be exploited to develop as a therapeutic target for breast cancer.

  9. MicroRNA-138 modulates metastasis and EMT in breast cancer cells by targeting vimentin.

    Science.gov (United States)

    Zhang, Jun; Liu, Dan; Feng, Zhuo; Mao, Jun; Zhang, Chunying; Lu, Ying; Li, Jiazhi; Zhang, Qingqing; Li, Qing; Li, Lianhong

    2016-02-01

    Increasing evidence indicates that dysregulation of microRNAs (miRNAs) plays critical roles in malignant transformation and tumor progression. In this study, in order to investigate the association of miR-138 with breast cancer we investigated the role of miR-138 in breast cancer metastasis. Levels of miR-138 were determined by qRT-PCR in 45 breast cancer samples. Cell migration and invasion assays were performed in a stably expressing miRNA-138 breast cancer cell line established using a lentivirus expression system. Epithelial-mesenchymal transition (EMT) was evaluated using qRT-PCR and Western Blots to detect epithelial marker E-cadherin and mesenchymal marker, vimentin. Luciferase reporter assays were used to identify downstream targets and biological function of miR-138. Breast cancer tissues had significantly lower expression of miR-138 compared to non-tumor tissues. Low miR-138 levels were associated with lymph node metastasis and invasion. miR-138 overexpression inhibited metastasis of breast cancer cells. miR-138 overexpression also down-regulated vimentin expression and upregulated E-cadherin expression, suggesting that miR-138 inhibited EMT. Our results support the involvement of miR-138 in breast tumorigenesis, especially lymph node metastasis. We propose that miR-138 might be used as therapeutic agent for breast cancer. Copyright © 2015. Published by Elsevier Masson SAS.

  10. In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-13-1-0184 TITLE: In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination PRINCIPAL...Sep 2013 - 14 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell...understand the early events that accompany invasive behavior in vivo , we proposed to develop a lineage-labeling system to detect and isolate cells of lung

  11. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    Science.gov (United States)

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

  12. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  13. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

    Science.gov (United States)

    Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. © 2014 AlphaMed Press.

  14. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    Science.gov (United States)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  15. Melanosome Motility in Fish Retinal Pigment Epithelial (RPE) Cells.

    Science.gov (United States)

    King-Smith, Christina

    2016-01-01

    Several model systems have been developed to investigate mechanism and regulation of intracellular organelle motility. The fish retinal pigment epithelial (RPE) cell represents a novel yet simple system for the study of organelle motility. Primary cultures of dissociated RPE cells are easily prepared and amenable to motility studies. In vivo, melanin-containing pigment granules (melanosomes) within fish RPE migrate distances up to 100 μm in response to light flux. When dissociated from the epithelial layer and cultured in vitro, RPE cells attach to the substrate with the apical projections extending radially from the central cell body. Melanosomes can be chemically triggered to aggregate or disperse throughout the projections, and are easily observed using phase contrast microscopy. Melanosome migration in RPE apical projections is dependent on actin filaments, and thus renders this model system useful for investigations of actin-dependent organelle motility.

  16. Metabotropic glutamate receptor 1 disrupts mammary acinar architecture and initiates malignant transformation of mammary epithelial cells.

    Science.gov (United States)

    Teh, Jessica L F; Shah, Raj; La Cava, Stephanie; Dolfi, Sonia C; Mehta, Madhura S; Kongara, Sameera; Price, Sandy; Ganesan, Shridar; Reuhl, Kenneth R; Hirshfield, Kim M; Karantza, Vassiliki; Chen, Suzie

    2015-05-01

    Metabotropic glutamate receptor 1 (mGluR1/Grm1) is a member of the G-protein-coupled receptor superfamily, which was once thought to only participate in synaptic transmission and neuronal excitability, but has more recently been implicated in non-neuronal tissue functions. We previously described the oncogenic properties of Grm1 in cultured melanocytes in vitro and in spontaneous melanoma development with 100 % penetrance in vivo. Aberrant mGluR1 expression was detected in 60-80 % of human melanoma cell lines and biopsy samples. As most human cancers are of epithelial origin, we utilized immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the transformative properties of Grm1. We introduced Grm1 into iMMECs and isolated several stable mGluR1-expressing clones. Phenotypic alterations in mammary acinar architecture were assessed using three-dimensional morphogenesis assays. We found that mGluR1-expressing iMMECs exhibited delayed lumen formation in association with decreased central acinar cell death, disrupted cell polarity, and a dramatic increase in the activation of the mitogen-activated protein kinase pathway. Orthotopic implantation of mGluR1-expressing iMMEC clones into mammary fat pads of immunodeficient nude mice resulted in mammary tumor formation in vivo. Persistent mGluR1 expression was required for the maintenance of the tumorigenic phenotypes in vitro and in vivo, as demonstrated by an inducible Grm1-silencing RNA system. Furthermore, mGluR1 was found be expressed in human breast cancer cell lines and breast tumor biopsies. Elevated levels of extracellular glutamate were observed in mGluR1-expressing breast cancer cell lines and concurrent treatment of MCF7 xenografts with glutamate release inhibitor, riluzole, and an AKT inhibitor led to suppression of tumor progression. Our results are likely relevant to human breast cancer, highlighting a putative role of mGluR1 in the pathophysiology of breast cancer and the potential

  17. File list: Oth.Lng.20.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Lng.20.AllAg.Tracheal_epithelial_cells hg19 TFs and others Lung Tracheal epithe...lial cells SRX268452,SRX268451,SRX268450 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Lng.20.AllAg.Tracheal_epithelial_cells.bed ...

  18. File list: Oth.Lng.05.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Lng.05.AllAg.Tracheal_epithelial_cells hg19 TFs and others Lung Tracheal epithe...lial cells SRX268452,SRX268450,SRX268451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Lng.05.AllAg.Tracheal_epithelial_cells.bed ...

  19. File list: Oth.Lng.10.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Lng.10.AllAg.Tracheal_epithelial_cells hg19 TFs and others Lung Tracheal epithe...lial cells SRX268452,SRX268450,SRX268451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Lng.10.AllAg.Tracheal_epithelial_cells.bed ...

  20. File list: DNS.Lng.20.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Lng.20.AllAg.Tracheal_epithelial_cells hg19 DNase-seq Lung Tracheal epithelial ...cells SRX1420085,SRX374730,SRX374729 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Lng.20.AllAg.Tracheal_epithelial_cells.bed ...

  1. File list: Unc.Lng.20.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Lng.20.AllAg.Tracheal_epithelial_cells hg19 Unclassified Lung Tracheal epitheli...al cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Lng.20.AllAg.Tracheal_epithelial_cells.bed ...

  2. File list: His.Lng.50.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Lng.50.AllAg.Tracheal_epithelial_cells hg19 Histone Lung Tracheal epithelial ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Lng.50.AllAg.Tracheal_epithelial_cells.bed ...

  3. File list: Unc.Lng.05.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Lng.05.AllAg.Tracheal_epithelial_cells hg19 Unclassified Lung Tracheal epitheli...al cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Lng.05.AllAg.Tracheal_epithelial_cells.bed ...

  4. In vitro co-culture of epithelial cells and smooth muscle cells on aligned nanofibrous scaffolds.

    Science.gov (United States)

    Kuppan, Purushothaman; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2017-12-01

    Esophagus is a complex, hollow organ consisting of epithelial cells in the inner mucosal layer and smooth muscle cells in the outer muscle layer. In the present study, we have evaluated the in vitro co-culture of epithelial cells and smooth muscle cells on the aligned nanofibrous scaffold made of PHBV, PHBV-gelatin, PCL and PCL-gelatin developed through electrospinning using rotating drum collector. Epithelial cells were labeled with cell tracker green while the smooth muscle cells were labeled with cell tracker red. Labeled cells were seeded on the aligned nanofibers matrices and tracked using laser scanning confocal microscopy. The results demonstrate that both epithelial and smooth muscle cells attach, extend, and proliferate over these nanofibrous matrices. Confocal z-sectioning shows that epithelial and smooth muscle cells tend to separate into two distinct layers on a single nanofiber system mimicking the in vivo anatomy. Cell viability assay showed that both types of cells are viable and also interact with each other. The functional gene expression of respective cell types demonstrates that both epithelial and smooth muscle cells are phenotypically as well as functionally active when they were co-cultured. Thus the study highlighted that aligned nanofibrous scaffolds could be potential alternative graft for esophageal tissue regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Immortalization protocols used in cell culture models of human breast morphogenesis

    DEFF Research Database (Denmark)

    Gudjonsson, T; Villadsen, R; Rønnov-Jessen, L

    2004-01-01

    cells enter an irreversible proliferation-arrested state referred to as replicative senescence. To overcome this obstacle for continuous long-term studies, replicative senescence can be bypassed by treatment of cells with chemical agents such as benzopyrene, by radiation or by transfection with viral...... breast cells in culture and optimizing a relevant microenvironment, which may help to define the niche that regulates breast differentiation and morphogenesis. In contrast to the general property of cancer, normal human cells have a finite lifespan. After a defined number of population doublings, normal...... of the tissue of origin. In recent years, we have sought to establish immortalized primary breast cells, which retain crucial characteristics of their original in situ tissue pattern. This review discusses various approaches to immortalization of breast-derived epithelial and stromal cells and the application...

  6. Smad2 overexpression enhances adhesion of gingival epithelial cells.

    Science.gov (United States)

    Hongo, Shoichi; Yamamoto, Tadashi; Yamashiro, Keisuke; Shimoe, Masayuki; Tomikawa, Kazuya; Ugawa, Yuki; Kochi, Shinsuke; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

    2016-11-01

    Gingival epithelial cells play an important role in preventing the initiation of periodontitis, by their hemidesmosomal adhesion to the tooth root surface. Adhesion requires integrin-extracellular matrix (ECM) interactions that are intricately regulated by transforming growth factor-β (TGF-β) signaling. However, the mechanisms underlying the interplay between adhesion molecules and TGF-β, especially the respective roles of Smad2 and Smad3, remain elusive. In this study, we examined the effects of Smad overexpression on gingival epithelial cell adhesion and expression profiles of integrin and ECM-related genes. Human gingival epithelial cells immortalized by the SV40 T-antigen were transfected with Smad2- and Smad3-overexpression vectors. A cell adhesion assay involving fluorescence detection of attached cells was performed using the ArrayScan imaging system. Real-time PCR was performed to examine the kinetics of integrin and ECM gene expression. In vitro and in vivo localization of adhesion molecules was examined by immunofluorescence analysis. By using SB431542, a specific inhibitor of the TGF-β type I receptor, Smad2/3 signaling was confirmed to be dominant in TGF-β1-induced cell adhesion. The Smad2-transfectant demonstrated higher potency for cell adhesion and integrin expression (α2, α5, β4, and β6) than the Smad3-transfectant, whereas little or no change in ECM expression was observed in either transfectant. Moreover, the gingival epithelium of transgenic mice that overexpressed Smad2 driven by the keratin 14 promoter showed increased integrin α2 expression. These findings indicate the crucial role of Smad2 in increased adhesion of gingival epithelial cells via upregulation of integrin α2. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Effect of TGF-β on ocular surface epithelial cells.

    Science.gov (United States)

    Benito, Maria Jesús; Calder, Virginia; Corrales, Rosa M; García-Vázquez, Carmen; Narayanan, Srihari; Herreras, José M; Stern, Michael E; Calonge, Margarita; Enríquez-de-Salamanca, Amalia

    2013-02-01

    A role for transforming growth factor (TGF)-β in the pathogenesis of some ocular surface diseases has been proposed. We determined if secretion of TGF-β and expression of TGF-β receptors RI, RII, and RIII by human ocular surface epithelial cells were modified under inflammatory conditions. We also determined how these cells responded to TGF-β. A human corneal epithelial (HCE) cell line and a conjunctival epithelial cell line (IOBA-NHC) were exposed to TGF-β1 and -β2 and to proinflammatory cytokines. TGF-β receptor mRNAs were analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) in both cell lines, and in conjunctival, limbal, and corneal epithelial cells from post-mortem human specimens. Expression of TGF-β receptors and pSMAD2/SMAD2 were determined by Western blot and immunofluorescence assays. Secretion of TGF-β isoforms, cytokine/chemokine, and metalloproteinases (MMPs) were analyzed in cell supernatants by immunobead-based assays. Secretory leukocyte proteinase inhibitor (SLPI) secretion was analyzed by enzyme-linked immunosorbent assay. TGF-β isoform and receptor gene expression was determined by RT-PCR in conjunctival epithelium of dry eye (DE) patients and healthy subjects. Our results showed that TGF-β RI expression was down-regulated with IL-4 exposure, whereas TGF-β RII and TGF-β2 were upregulated by TNF-α in HCE cells. TGF-β RIII receptor expression was upregulated in IOBA-NHC cells by TNF-α and IFN-γ. SMAD2 phosphorylation occurred in HCE and IOBA-NHC cells after TGF-β treatment. TGF-β significantly up- and down-regulated secretion of several cytokines/chemokines by both cell lines and MMP by HCE cells. TGF-β2 and TGF-β3 were upregulated and TGF-β RIII mRNA was down-regulated in DE conjunctival epithelium. These results show that TGF-β plays an important role in directing local inflammatory responses in ocular surface epithelial cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Effect of infertility treatment and pregnancy-related hormones on breast cell proliferation in vitro.

    Science.gov (United States)

    Cooley, Anne; Matthews, Laura; Zelivianski, Stanislav; Hardy, Ashley; Jeruss, Jacqueline S

    2012-01-01

    Breast cancer development involves a series of mutations in a heterogeneous group of proto-oncogenes/tumor suppressor genes that alter mammary cells to create a microenvironment permissive to tumorigenesis. Exposure to hormones during infertility treatment may have a mutagenic effect on normal mammary epithelial cells, high-risk breast lesions and early-stage breast cancers. Our goal was to understand the association between infertility treatment and normal and cancerous breast cell proliferation. MCF-10A normal mammary cells and the breast cancer cell lines MCF-7 [estrogen receptor (ER)-positive, well differentiated] and HCC 1937 (ER-negative, aggressive, BRCA1 mutation) were treated with the weak ER activator clomiphene citrate and hormones that are increased during infertility treatment. Direct effects of treatment on cell proliferation and colony growth were determined. While clomiphene citrate had no effect on MCF-10A cells or MCF-7 breast cancer cells, it decreased proliferation of HCC 1937 versus untreated cells (P= 0.003). Estrogen had no effect on either MCF-10A or HCC 1937 cells but, as expected, increased cell proliferation (20-100 nM; P≤0.002) and colony growth (10-30 nM; Pinfertility regimens on ER-positive breast cancer cells and validate the potential protective effect of pregnancy-related exposure to hCG.

  9. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Doupnik, C.A.; Leikauf, G.D. (Univ. of Cincinnati College of Medicine, OH (USA))

    1990-10-01

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with (3H)arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. (3H)arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein.

  10. Reduced uptake of the proliferation-seeking radiotracer technetium-99m-labelled pentavalent dimercaptosuccinic acid in a 47-year-old woman with severe breast epithelial hyperplasia taking ibuprofen: a case report

    Directory of Open Access Journals (Sweden)

    Marinopoulos Spyridon G

    2010-03-01

    Full Text Available Abstract Introduction Recent studies have reported a risk reduction in the progression of benign breast disease to breast carcinoma through COX-2 pathways. Case presentation We present a case of severe epithelial hyperplasia in a 47-year-old woman with increased breast density submitted to scintimammography by the proliferation-imaging tracer Technetium-99m-labelled pentavalent dimercaptosuccinic acid, before and after an oral ibuprofen treatment for 4 weeks. The radiotracer uptake after ibuprofen intake was significantly reduced, both visually and by semi-quantitative analysis, based on a calculation of lesion-to-background ratios. Conclusion In proliferating breast lesions, scintigraphically displayed reduction in Technetium-99m-labelled pentavalent dimercaptosuccinic acid uptake may indicate inhibition by ibuprofen in the pathway of malignant epithelial cell transformation.

  11. Selective release of microRNA species from normal and malignant mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lucy Pigati

    2010-10-01

    Full Text Available MicroRNAs (miRNAs in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

  12. Characterization of a naturally occurring breast cancer subset enriched in EMT and stem cell characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Hennessy, Bryan T.; Gonzalez-Angulo, Ana-Maria; Stemke-Hale, Katherine; Gilcrease, Michael Z.; Krishnamurthy, Savitri; Lee, Ju-Seog; Fridlyand, Jane; Sahin, Aysegul; Agarwal, Roshan; Joy, Corwin; Liu, Wenbin; Stivers, David; Baggerly, Keith; Carey, Mark; Lluch, Ana; Monteagudo, Carlos; He, Xiaping; Weigman, Victor; Fan, Cheng; Palazzo, Juan; Hortobagyi, Gabriel N.; Nolden, Laura K.; Wang, Nicholas J.; Valero, Vicente; Gray, Joe W.; Perou, Charles M.; Mills, Gordon B.

    2009-05-19

    Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor-positive cancers (34.5%; P = 0.32), 17 of 75 HER-2-positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a 'tumorigenic' signature defined using CD44{sup +}/CD24{sup -} breast tumor-initiating stem cell-like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell-like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets.

  13. Estradiol increases mucus synthesis in bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Anthony Tam

    Full Text Available Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI. Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0 cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6 mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.

  14. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  15. Alveolar epithelial type II cell: defender of the alveolus revisited

    Directory of Open Access Journals (Sweden)

    Fehrenbach Heinz

    2001-01-01

    Full Text Available Abstract In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2 cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today.

  16. Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Satoru Yoshida

    Full Text Available BACKGROUND: Application of induced pluripotent stem (iPS cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. METHODOLOGY/PRINCIPAL FINDINGS: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. CONCLUSIONS/SIGNIFICANCE: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.

  17. Generation of Stratified Squamous Epithelial Progenitor Cells from Mouse Induced Pluripotent Stem Cells

    Science.gov (United States)

    Yoshida, Satoru; Yasuda, Miyuki; Miyashita, Hideyuki; Ogawa, Yoko; Yoshida, Tetsu; Matsuzaki, Yumi; Tsubota, Kazuo; Okano, Hideyuki; Shimmura, Shigeto

    2011-01-01

    Background Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. Methodology/Principal Findings We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. Conclusions/Significance These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets. PMID:22174914

  18. Efficient and simple approach to in vitro culture of primary epithelial cancer cells.

    Science.gov (United States)

    Janik, Karolina; Popeda, Marta; Peciak, Joanna; Rosiak, Kamila; Smolarz, Maciej; Treda, Cezary; Rieske, Piotr; Stoczynska-Fidelus, Ewelina; Ksiazkiewicz, Magdalena

    2016-12-01

    Primary cancer cells constitute a favourable testing platform for in vitro research in oncology field as they reflect tumour state more accurately than the most commonly employed stable cell lines. Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice.We have compared protocols for primary cultures, differing in media components and plate coatings. In terms of culture establishment, application of Geltrex® coating demonstrated equal efficiency to feeder layer (83% compared with 72% successfully established breast and 80% compared with 80% prostate tumour specimens), yet it was substantially less complicated and easier to validate. Both Geltrex® coating and tissue-specific primary cell medium were permanently required to successfully maintain primary epithelial prostate cancer cells (PEPCs) in culture. In case of primary epithelial breast cancer cells (PEBCs), collagen I coating enabled to obtain comparable number of passages to Geltrex® coating (P=0.438). Commercial primary cell media demonstrated lower efficiency than tissue-specific ones (PEPCs-5 compared with 8 and PEBCs-6 compared with 9 passages). Interestingly, both analysed tumour types were unsusceptible to induction of culture lifespan extension when transduced with SV40LT, BMI-1 or hEST2 genes, commonly applied as potential immortalizing agents.In conclusion, the approach based on extracellular matrix reconstitution and tissue-specific primary cell media is easy to validate and provides in vitro expansion sufficient for analytical purposes (approximately 8 passages). Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses. © 2016 The Author(s).

  19. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics.

    Directory of Open Access Journals (Sweden)

    Cornelia Blume

    Full Text Available The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.

  20. Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells.

    Science.gov (United States)

    Nagahara, Teruya; Shiraha, Hidenori; Sawahara, Hiroaki; Uchida, Daisuke; Takeuchi, Yasuto; Iwamuro, Masaya; Kataoka, Junro; Horiguchi, Shigeru; Kuwaki, Takeshi; Onishi, Hideki; Nakamura, Shinichiro; Takaki, Akinobu; Nouso, Kazuhiro; Yamamoto, Kazuhide

    2015-09-01

    Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA‑treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1

  1. The role of Vimentin in Regulating Cell Invasive Migration in Dense Cultures of Breast Carcinoma Cells

    Science.gov (United States)

    Messica, Yonatan; Laser-Azogui, Adi; Volberg, Tova; Elisha, Yair; Lysakovskaia, Kseniia; Eils, Roland; Gladilin, Evgeny; Geiger, Benjamin; Beck, Roy

    2017-11-01

    Cell migration and mechanics are tightly regulated by the integrated activities of the various cytoskeletal networks. In cancer cells, cytoskeletal modulations have been implicated in the loss of tissue integrity, and acquisition of an invasive phenotype. In epithelial cancers, for example, increased expression of the cytoskeletal filament protein vimentin correlates with metastatic potential. Nonetheless, the exact mechanism whereby vimentin affects cell motility remains poorly understood. In this study, we measured the effects of vimentin expression on the mechano-elastic and migratory properties of the highly invasive breast carcinoma cell line MDA231. We demonstrate here that vimentin stiffens cells and enhances cell migration in dense cultures, but exerts little or no effect on the migration of sparsely plated cells. These results suggest that cell-cell interactions play a key role in regulating cell migration, and coordinating cell movement in dense cultures. Our findings pave the way towards understanding the relationship between cell migration and mechanics, in a biologically relevant context.

  2. 2D Difference Gel Electrophoresis Analysis of Different Time Points during the Course of Neoplastic Transformation of Human Mammary Epithelial Cells

    OpenAIRE

    DeAngelis, J. Tyson; Li, Yuanyuan; Mitchell, Natalie; Wilson, Landon; Kim, Helen; Tollefsbol, Trygve O.

    2010-01-01

    Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesi...

  3. Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

    Science.gov (United States)

    Herceg, Zdenko

    Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the

  4. Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

    Energy Technology Data Exchange (ETDEWEB)

    Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.; Onishi,Akiko; Campbell, Kevin P.; Bissell, Mina J.; Muschler, John L.

    2006-02-17

    Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.

  5. Small molecule mediated proliferation of primary retinal pigment epithelial cells.

    Science.gov (United States)

    Swoboda, Jonathan G; Elliott, Jimmy; Deshmukh, Vishal; de Lichtervelde, Lorenzo; Shen, Weijun; Tremblay, Matthew S; Peters, Eric C; Cho, Charles Y; Lu, Bin; Girman, Sergej; Wang, Shaomei; Schultz, Peter G

    2013-07-19

    Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for transplantation. Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.

  6. ADP-ribosylation factor 1 expression regulates epithelial-mesenchymal transition and predicts poor clinical outcome in triple-negative breast cancer

    Science.gov (United States)

    Schlienger, Sabrina; Campbell, Shirley; Pasquin, Sarah; Gaboury, Louis; Claing, Audrey

    2016-01-01

    Metastatic capacities are fundamental features of tumor malignancy. ADP-ribosylation factor (ARF) 1 has emerged as a key regulator of invasion in breast cancer cells. However, the importance of this GTPase, in vivo, remains to be demonstrated. We report that ARF1 is highly expressed in breast tumors of the most aggressive and advanced subtypes. Furthermore, we show that lowered expression of ARF1 impairs growth of primary tumors and inhibits lung metastasis in a murine xenograft model. To understand how ARF1 contributes to invasiveness, we used a poorly invasive breast cancer cell line, MCF7 (ER+), and examined the effects of overexpressing ARF1 to levels similar to that found in invasive cell lines. We demonstrate that ARF1 overexpression leads to the epithelial-mesenchymal transition (EMT). Mechanistically, ARF1 controls cell–cell adhesion through ß-catenin and E-cadherin, oncogenic Ras activation and expression of EMT inducers. We further show that ARF1 overexpression enhances invasion, proliferation and resistance to a chemotherapeutic agent. In vivo, ARF1 overexpressing MCF7 cells are able to form more metastases to the lung. Overall, our findings demonstrate that ARF1 is a molecular switch for cancer progression and thus suggest that limiting the expression/activation of this GTPase could help improve outcome for breast cancer patients. PMID:26908458

  7. Osmoregulation of chloride channels in epithelial cells

    NARCIS (Netherlands)

    C.H. Lim (Christina)

    2008-01-01

    markdownabstract__Abstract__ The plasma membrane of mammalian cells is formed by two layers of lipids (lipid bilayer), primarily phospholipids, glycolipids and cholesterol, in which many different proteins are embedded. Phospholipid consists of a glycerol backbone esterified to fatty acids

  8. Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions

    National Research Council Canada - National Science Library

    Peinado, Hector; Quintanilla, Miguel; Cano, Amparo

    2003-01-01

    .... We show here that transforming growth factor beta-1 (TGFbeta1) induces Snail expression in Madin-Darby canine kidney cells and triggers epithelial-mesenchymal transitions by a mechanism dependent on the MAPK signaling pathway...

  9. Current Concepts and Occurrence of Epithelial Odontogenic Tumors: II. Calcifying Epithelial Odontogenic Tumor Versus Ghost Cell Odontogenic Tumors Derived from Calcifying Odontogenic Cyst

    OpenAIRE

    Lee, Suk Keun; Kim, Yeon Sook

    2014-01-01

    Calcifying epithelial odontogenic tumors (CEOTs) and ghost cell odontogenic tumors (GCOTs) are characteristic odontogenic origin epithelial tumors which produce calcifying materials from transformed epithelial tumor cells. CEOT is a benign odontogenic tumor composed of polygonal epithelial tumor cells that show retrogressive calcific changes, amyloid-like deposition, and clear cytoplasm. Differentially, GCOTs are a group of transient tumors characterized by ghost cell presence, which comprise...

  10. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Directory of Open Access Journals (Sweden)

    Ivana Viktorinová

    2017-11-01

    Full Text Available Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  11. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Science.gov (United States)

    Viktorinová, Ivana; Henry, Ian; Tomancak, Pavel

    2017-11-01

    Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  12. The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures

    Directory of Open Access Journals (Sweden)

    Soto Ana M

    2010-06-01

    Full Text Available Abstract Background Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. Methods The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks, the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. Results Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. Conclusions Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to

  13. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration.

    Science.gov (United States)

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-10-13

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway.

  14. Serratia marcescens is injurious to intestinal epithelial cells.

    Science.gov (United States)

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

  15. Molecular cloning, sequence analysis, and function of the intestinal epithelial stem cell marker Bmi1 in pig intestinal epithelial cells.

    Science.gov (United States)

    Li, C-M; Yan, H-C; Fu, H-L; Xu, G-F; Wang, X-Q

    2014-01-01

    In the present work, we cloned the full-length cDNA of the pig Bmi1 gene (BMI1 polycomb ring finger oncogene), which has been indicated as an intestinal epithelial stem cell (IESC) marker in other mammals. This paper provides the first report of the function of Bmi1 in pig intestinal epithelial cells and a brief description of its underlying mechanism. Rapid amplification of cDNA ends technology was used to clone the complete pig Bmi1 sequence, and a Bmi1-pcDNA3.1 vector was constructed for transfection into an intestinal porcine epithelial cell line (IPEC-1). The proliferation ability of the cells was estimated using the MTT assay and the EdU incorporation method at different time points after seeding. Cell cycle information was detected by flow cytometry. The mRNA abundances of cell cycle-related genes were also measured. The results indicated that the pig Bmi1 cDNA is 3,193 bp in length and consists of a 981 bp open reading frame, a 256 bp 5´ untranslated region (UTR), and a 1,956 bp 3' UTR. The transcript contains no signal peptides, and there are no transmembrane regions in the pig Bmi1 coded protein, which has a total of 326 AA. The overexpression of the pig Bmi1 in the IPEC-1 cells led to increased cell proliferation and a lower percentage of cells in the G1 and S phases (P cells in the G2 phase (P 0.05). Our data suggested that pig Bmi1 can increase the proliferation of IPEC-1 cells by promoting the G1/S transition and the overall cell cycle process.

  16. Tumor associated macrophage × cancer cell hybrids may acquire cancer stem cell properties in breast cancer.

    Directory of Open Access Journals (Sweden)

    Jingxian Ding

    Full Text Available Breast cancer is one of the most frequently diagnosed cancers among women, and metastasis makes it lethal. Tumor-associated macrophages (TAMs that acquire an alternatively activated macrophage (M2 phenotype may promote metastasis. However, the underlying mechanisms are still elusive. Here, we examined how TAMs interact with breast cancer cells to promote metastasis. Immunohistochemistry was used to examine the expression of the M2-specific antigen CD163 in paraffin-embedded mammary carcinoma blocks to explore fusion events in breast cancer patients. U937 cells were used as a substitute for human monocytes, and these cells differentiated into M2 macrophages following phorbol 12-myristate 13-acetate (PMA and M-CSF stimulation. M2 macrophages and the breast cancer cell lines MCF-7 and MDA-MB-231 fused in the presence of 50% polyethylene glycol. Hybrids were isolated by fluorescence-activated cell sorting, and the relevant cell biological properties were compared with their parental counterparts. Breast cancer stem cell (BCSC-related markers were quantified by immunofluorescence staining, RT-PCR, quantitative RT-PCR and/or western blotting. The tumor-initiating and metastatic capacities of the hybrids and their parental counterparts were assessed in NOD/SCID mice. We found that the CD163 expression rate in breast cancer tissues varied significantly and correlated with estrogen receptor status (p0.05. Characterization of the fusion hybrids revealed a more aggressive phenotype, including increased migration, invasion and tumorigenicity, but reduced proliferative ability, compared with the parental lines. The hybrids also gained a CD44(+CD24(-/low phenotype and over-expressed epithelial-mesenchymal transition-associated genes. These results indicate that TAMs may promote breast cancer metastasis through cell fusion, and the hybrids may gain a BCSC phenotype.

  17. Protective Effects of Trehalose on the Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Pasquale Aragona

    2014-01-01

    Full Text Available Purpose. Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Methods. Twelve patients undergoing laser subepithelial keratomileusis (LASEK were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. Results. In both trehalose-untreated eyes (TUE and trehalose-treated eyes (TTE, the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Conclusions. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

  18. The origin of stroma surrounding epithelial ovarian cancer cells.

    Science.gov (United States)

    Akahane, Tomoko; Hirasawa, Akira; Tsuda, Hiroshi; Kataoka, Fumio; Nishimura, Sadako; Tanaka, Hideo; Tominaga, Eiichiro; Nomura, Hiroyuki; Chiyoda, Tatsuyuki; Iguchi, Yoko; Yamagami, Wataru; Susumu, Nobuyuki; Aoki, Daisuke

    2013-01-01

    Cancer stroma is thought to play an important role in tumor behavior, including invasion or metastasis and response to therapy. Cancer stroma is generally thought either to be non-neoplastic cells, including tissue-marrow or bone-marrow-derived fibroblasts, or to originate in epithelial mesenchymal transition of cancer cells. In this study, we evaluated the status of the p53 gene in both the cancer cells and the cancer stroma in epithelial ovarian cancer (EOC) to elucidate the origin of the stroma. Samples from 16 EOC patients were included in this study. Tumor cells and adjacent nontumor stromal cells were microdissected and DNA was extracted separately. We analyzed p53 sequences (exons 5-8) of both cancer and stromal tissues in all cases. Furthermore, we examined p53 protein expression in all cases. Mutations in p53 were detected in 9 of the 16 EOCs: in 8 of these cases, the mutations were detected only in cancer cells. In 1 case, the same mutation (R248Q) was detected in both cancer and stromal tissues, and p53 protein expression was detected in both the cancer cells and the cancer stroma. Most cancer stroma in EOC is thought to originate from non-neoplastic cells, but some parts of the cancer stroma might originate from cancer cells.

  19. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    Science.gov (United States)

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  20. Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

    Science.gov (United States)

    Miyata, Ryo; Nomura, Kazuaki; Kakuki, Takuya; Takano, Ken-Ichi; Kohno, Takayuki; Konno, Takumi; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

    2015-04-01

    The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

  1. Toca-1 is suppressed by p53 to limit breast cancer cell invasion and tumor metastasis.

    Science.gov (United States)

    Chander, Harish; Brien, Colin D; Truesdell, Peter; Watt, Kathleen; Meens, Jalna; Schick, Colleen; Germain, Doris; Craig, Andrew W B

    2014-12-30

    Transducer of Cdc42-dependent actin assembly-1 (Toca-1) recruits actin regulatory proteins to invadopodia, and promotes breast tumor metastasis. Since metastatic breast tumors frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression. Normal mammary epithelial cells (HBL-100, MCF10A) and breast cancer cell lines expressing wild-type (WT) p53 (DU4475, MTLn3) were treated with camptothecin or Nutlin-3 to stabilize p53 to test effects on Toca-1 mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assays were performed to identify p53 binding site in Toca-1 gene. Stable silencing of p53 and Toca-1 were performed in MTLn3 cells to test effects on invadopodia and cell invasion in vitro, and tumor metastasis in vivo. We observed that breast cancer cell lines with mutant p53 have high levels of Toca-1 compared to those with WT p53. Stabilization of WT p53 led to further reduction in Toca-1 mRNA and protein levels in normal breast epithelial cells and breast cancer cells. ChIP assays revealed p53 binding within intron 2 of toca1, and reduced histone acetylation within its promoter region upon p53 upregulation or activation. Stable silencing of WT p53 in MTLn3 cells led to increased extracellular matrix degradation and cell invasion compared to control cells. Interestingly, the combined silencing of p53 and Toca-1 led to a partial rescue of these effects of p53 silencing in vitro and reduced lung metastases in mice. In human breast tumors, Toca-1 levels were high in subtypes with frequent p53 mutations, and high Toca-1 transcript levels correlated with increased risk of relapse. Based on these findings, we conclude that loss of p53 tumor suppressor function in breast cancers leads to upregulation of Toca-1, and results in enhanced risk of developing metastatic disease.

  2. Advances in Classification and Research Methods of Lung Epithelial Stem 
and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Minhua DENG

    2017-02-01

    Full Text Available Isolation and characterization of lung epithelial stem and progenitor cells and understanding of their specific role in lung physiopathology are critical for preventing and controlling lung diseases including lung cancer. In this review, we summarized recent advances in classification and research methods of lung epithelial stem and progenitor cells. Lung epithelial stem and progenitor cells were region-specific, which primarily included basal cells and duct cells in proximal airway, Clara cells, variant Clara cells, bronchioalveolar stem cells and induced krt5+ cells in bronchioles, type II alveolar cells and type II alveolar progenitor cells in alveoli. The research methods of lung epithelial stem and progenitor cells were mainly focused on lung injury models, lineage-tracing experiments, three dimensional culture, transplantation, chronic labeled cells and single-cell transcriptome analysis. Lastly, the potential relationship between lung epithelial stem and progenitor cells and lung cancer as well as lung cancer stem cell-targeted drug development were briefly reviewed.

  3. [Advances in Classification and Research Methods of Lung Epithelial Stem 
and Progenitor Cells].

    Science.gov (United States)

    Deng, Minhua; Li, Jinhua; Gan, Ye; Chen, Ping

    2017-02-20

    Isolation and characterization of lung epithelial stem and progenitor cells and understanding of their specific role in lung physiopathology are critical for preventing and controlling lung diseases including lung cancer. In this review, we summarized recent advances in classification and research methods of lung epithelial stem and progenitor cells. Lung epithelial stem and progenitor cells were region-specific, which primarily included basal cells and duct cells in proximal airway, Clara cells, variant Clara cells, bronchioalveolar stem cells and induced krt5+ cells in bronchioles, type II alveolar cells and type II alveolar progenitor cells in alveoli. The research methods of lung epithelial stem and progenitor cells were mainly focused on lung injury models, lineage-tracing experiments, three dimensional culture, transplantation, chronic labeled cells and single-cell transcriptome analysis. Lastly, the potential relationship between lung epithelial stem and progenitor cells and lung cancer as well as lung cancer stem cell-targeted drug development were briefly reviewed.

  4. Primary mouse small intestinal epithelial cell cultures

    NARCIS (Netherlands)

    Sato, T.; Clevers, H.

    2013-01-01

    The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have

  5. [Adhesion of clinical Candida albicans isolate to buccal epithelial cells].

    Science.gov (United States)

    Wellmer, A

    1999-01-01

    Mucosal adherence and germ tube formation are considered to be important virulence factors of C. albicans. Adherence is a precondition for colonisation and invasion. We investigated 11 clinical isolates (among them 5 cases recovered from oesophageal thrush) for quantification of the two characteristics and correlated the results with clinical data. Adherence was measured on buccal epithelial cells and the continuous flow culture was used for quantification of germ tube formation. Adherence of strains recovered from clinically, culturally and serologically confirmed oesophageal thrush adhered stronger to buccal epithelial cells than isolates from patients with heavy colonisation without signs of candidosis. Strains with stronger adherence showed a significantly faster and an increased germ tube formation in the continuous flow culture. Strains from oesophageal thrush therefore show a more marked expression of the investigated virulence factors. Therefore a good adherence is a necessity for infection of the oesophagus by C. albicans. The preferential isolation of C. albicans from oesophageal thrush (> 90%) supports this assumption.

  6. Interaction between submicron COD crystals and renal epithelial cells.

    Science.gov (United States)

    Peng, Hua; Ouyang, Jian-Ming; Yao, Xiu-Qiong; Yang, Ru-E

    2012-01-01

    This study aims to investigate the adhesion characteristics between submicron calcium oxalate dihydrate (COD) with a size of 150 ± 50 nm and African green monkey kidney epithelial cells (Vero cells) before and after damage, and to discuss the mechanism of kidney stone formation. Vero cells were oxidatively injured by hydrogen peroxide to establish a model of injured cells. Scanning electron microscopy was used to observe Vero-COD adhesion. Inductively coupled plasma emission spectrometry was used to quantitatively measure the amount of adhered COD microcrystals. Nanoparticle size analyzer and laser scanning confocal microscopy were performed to measure the change in the zeta potential on the Vero cell surface and the change in osteopontin expression during the adhesion process, respectively. The level of cell injury was evaluated by measuring the changes in malonaldehyde content, and cell viability during the adhesion process. The adhesion capacity of Vero cells in the injury group to COD microcrystals was obviously stronger than that of Vero cells in the control group. After adhesion to COD, cell viability dropped, both malonaldehyde content and cell surface zeta potential increased, and the fluorescence intensity of osteopontin decreased because the osteopontin molecules were successfully covered by COD. Submicron COD further damaged the cells during the adhesion process, especially for Vero cells in the control group, leading to an elevated amount of attached microcrystals. Submicron COD can further damage injured Vero cells during the adhesion process. The amount of attached microcrystals is proportional to the degree of cell damage. The increased amount of microcrystals that adhered to the injured epithelial cells plays an important role in the formation of early-stage kidney stones.

  7. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine

    NARCIS (Netherlands)

    Klunder, Leon J.; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C. D.

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we

  8. Age and the means of bypassing stasis are determinants of the intrinsic subtypes of immortalized human mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Jonathan K Lee

    2015-03-01

    Full Text Available Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis are independent determinants of subtype in immortalized human

  9. Ivermectin inhibits growth of Chlamydia trachomatis in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Matthew A Pettengill

    Full Text Available Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia.

  10. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob

    2003-01-01

    Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers...... of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium....

  11. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducibl...... nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells....

  12. The role of diet in the development of breast cancer: a case-control study of patients with breast cancer, benign epithelial hyperplasia and fibrocystic disease of the breast.

    Science.gov (United States)

    Ingram, D. M.; Nottage, E.; Roberts, T.

    1991-01-01

    A case-control study was undertaken to investigate the role of diet in women with breast cancer, and in two groups of women with benign breast disease: epithelial hyperplasia, and fibrocystic disease without hyperplasia. The study provides data which suggest that the consumption of red meat, savoury meals (pizza, pies, stew, etc.) and of starches is disadvantageous, while the consumption of chicken and fish, and of fruit, appears to be beneficial. These patterns were present for both the breast cancer patients and the patients with benign epithelial hyperplasia. One-third of breast cancer patients had changed their diet after their diagnosis, compared to only around 12% in controls and patients with benign breast disease. Overall, the women studied had changed their diet to reduce their intake of sugars, dairy products and meat, and increased their intake of poultry, fish, fruit and vegetables over the past decade; but the breast cancer group was less likely to have made this change. PMID:1854621

  13. Stretch induces cytokine release by alveolar epithelial cells in vitro.

    Science.gov (United States)

    Vlahakis, N E; Schroeder, M A; Limper, A H; Hubmayr, R D

    1999-07-01

    Mechanical ventilation can injure the lung, causing edema and alveolar inflammation. Interleukin-8 (IL-8) plays an important role in this inflammatory response. We postulated that cyclic cell stretch upregulates the production and release of IL-8 by human alveolar epithelium in the absence of structural cell damage or paracrine stimulation. To test this hypothesis, alveolar epithelial cells (A549 cells) were cultured on a deformable silicoelastic membrane. When stretched by 30% for up to 48 h, the cells released 49 +/- 34% more IL-8 (P static controls. Smaller deformations (20% stretch) produced no consistent increase in IL-8. Stretch of 4 h duration increased IL-8 gene transcription fourfold above baseline. Stretch had no effect on cell proliferation, cell viability as assessed by (51)Cr release assay, or the release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha. We conclude that deformation per se can trigger inflammatory signaling and that alveolar epithelial cells may be active participants in the alveolitis associated with ventilator-induced lung injury.

  14. Cisplatin induces differentiation of breast cancer cells.

    Science.gov (United States)

    Prabhakaran, Praseetha; Hassiotou, Foteini; Blancafort, Pilar; Filgueira, Luis

    2013-01-01

    Breast tumors are heterogeneous including cells with stem cell properties and more differentiated cells. This heterogeneity is reflected into the molecular breast cancer subtypes. Breast cancer stem cells are resistant to chemotherapy, thus recent efforts are focusing on identifying treatments that shift them toward a more differentiated phenotype, making them more susceptible to chemotherapy. We examined whether the drug cisplatin induces differentiation in breast cancer cell lines that represent different breast cancer subtypes. We used three cell lines representing triple-negative breast cancers, BT-549 and MDA-MB-231 (claudin-low), and MDA-MB-468 (basal-like), along with estrogen and progesterone receptor positive MCF-7 cells (luminal). Cisplatin was applied at 2.5, 5, 10, and 20 μM, and cell viability and proliferation were measured using MTS and BrdU assays, respectively. The effect of cisplatin on the cellular hierarchy was examined by flow cytometry, immunofluorescence and qRT-PCR. Cisplatin treatment of 10 and 20 μM reduced cell viability by 36-51% and proliferation capacity by 36-67%. Treatment with cisplatin resulted in 12-67% down-regulation of stem cell markers (CD49f, SSEA4) and 10-130% up-regulation of differentiation markers (CK18, SMA, β-tubulin). At the mRNA level, CD49f was down-regulated whilst β-tubulin was up-regulated in the claudin-low cell lines. SSEA4 protein expression decreased upon cisplatin treatment, but SSEA4 mRNA expression increased indicating a differential regulation of cisplatin at the post-transcriptional level. It is concluded that cisplatin reduces breast cancer cell survival and induces differentiation of stem/progenitor cell subpopulations within breast cancer cell lines. These effects indicate the potential of this drug to target specific chemotherapy-resistant cells within a tumor.

  15. Isolation, separation, and characterization of epithelial and connective cells from rat palate

    Energy Technology Data Exchange (ETDEWEB)

    Terranova, Victor Paul [Univ. of Rochester, NY (United States)

    1979-01-01

    Epithelial and connective tissue cells were isolated from rat palate by sequential collagenase, hyaluronidase and trypsin digestion of the extracellular matrix. Differences between the two populations were noted with respect to total cell protein, total cell water, proline uptake and incorporation, percent collagen synthesized, effects of parathyroid hormone, metabolism of D-valine and cell density. Basal epithelial cells were subsequently separated from the heterogeneous epithelial cell population on shallow linear density gradients by velocity centrifugation. The type of collagen synthesized by the basal epithelial cells was compared to the type of collagen synthesized by the connective tissue cells by means of labeled amino acid incorporation ratios. Cells isolated from the epithelial and connective tissue were compared. From these studies it can be concluded that epithelial and connective tissue cells can be isolated from rat palate as viable and distinct populations with respect to the biochemical parameters examined. Furthermore, subpopulations can be separated and biochemically characterized.

  16. Airway Epithelial Cell Cilia and Obstructive Lung Disease

    Directory of Open Access Journals (Sweden)

    Asma Yaghi

    2016-11-01

    Full Text Available Airway epithelium is the first line of defense against exposure of the airway and lung to various inflammatory stimuli. Ciliary beating of airway epithelial cells constitutes an important part of the mucociliary transport apparatus. To be effective in transporting secretions out of the lung, the mucociliary transport apparatus must exhibit a cohesive beating of all ciliated epithelial cells that line the upper and lower respiratory tract. Cilia function can be modulated by exposures to endogenous and exogenous factors and by the viscosity of the mucus lining the epithelium. Cilia function is impaired in lung diseases such as COPD and asthma, and pharmacologic agents can modulate cilia function and mucus viscosity. Cilia beating is reduced in COPD, however, more research is needed to determine the structural-functional regulation of ciliary beating via all signaling pathways and how this might relate to the initiation or progression of obstructive lung diseases. Additionally, genotypes and how these can influence phenotypes and epithelial cell cilia function and structure should be taken into consideration in future investigations.

  17. Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy.

    Science.gov (United States)

    Ghosh, Moumita; Ahmad, Shama; White, Carl W; Reynolds, Susan D

    2017-01-01

    Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.

  18. Early Detection of Breast Cancer Using Molecular Beacons

    National Research Council Canada - National Science Library

    Yang, Lily

    2008-01-01

    .... We proposed to use molecular beacon technology to detect the level of expression of several biomarker genes that are highly expressed in breast cancer cells but not in normal breast epithelial cells...

  19. Epithelial cell adhesion molecule-1 (ECAM1) is required in the maintenance of corneal epithelial barrier integrity.

    Science.gov (United States)

    Zhou, Jinzi; Jiang, Jian; Wang, Shuhong; Xia, Xiaobo

    2016-01-01

    Corneal epithelial barrier integrity is critical in the maintenance of the corneal homeostasis. The corneal barrier dysfunction may be associated with the pathogenesis of a number of eye diseases. In this study, we assessed the expression of epithelial cell adhesion molecule-1 (ECAM1) in human corneal epithelial cells (HCE). The epithelial barrier function of the corneal epithelial monolayer was determined in Transwells. We found that the HCE cells expressed ECAM1. Knockdown of ECAM1 markedly compromised the HCE monolayer barrier function. A complex of ECAM1, claudin1, and occludin was detected in the HCE monolayers, which was not detected in the ECAM1-null HCE monolayers. Exposure to the proinflammatory cytokine, interleukin-13, inhibited the expression of ECAM1 in HCE cells and compromised the barrier function, which was prevented in the HCE monolayer with the ECAM1 overexpression. In conclusion, ECAM1 is required in the formation of the tight junction complex and maintaining the corneal epithelial barrier function. © 2015 International Federation for Cell Biology.

  20. Human breast microvascular endothelial cells retain phenotypic traits in long-term finite life span culture

    DEFF Research Database (Denmark)

    Sigurdsson, Valgardur; Fridriksdottir, Agla J R; Kjartansson, Jens

    2007-01-01

    uptake of low-density lipoprotein, and had E-selectin induced upon treatment with tumor necrosis factor-alpha. The first signs of senescence in passage 14 were accompanied by gain of trisomy 11. At passage 18 cells showed chromosomal aberrations and growth arrest as revealed by beta......Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits......-galactosidase staining. We demonstrate here that breast microvasculature may serve as a large-scale source for expansion of BRENCs with molecular and functional traits preserved. These cells will form the basis for studies on the role of endothelial cells in breast morphogenesis....

  1. Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Aihua Hou

    Full Text Available PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement