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Sample records for ductal epithelial cells

  1. Imbalanced expression of Tif1γ inhibits pancreatic ductal epithelial cell growth

    PubMed Central

    Ligr, Martin; Wu, Xinyu; Daniels, Garrett; Zhang, David; Wang, Huamin; Hajdu, Cristina; Wang, Jinhua; Pan, Ruimin; Pei, Zhiheng; Zhang, Lanjing; Melis, Marcovalerio; Pincus, Matthew R; Saunders, John K; Lee, Peng; Xu, Ruliang

    2014-01-01

    Transcriptional intermediary factor 1 gamma (Tif1γ) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGFβ pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1γ interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1γ inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1γ and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1γ resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1γ resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1γ reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1γ results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1γ may inhibit tumor invasion. These data suggest that Tif1γ might serve as a potential therapeutic target for pancreatic cancer. PMID:24959375

  2. KrasG12D induces EGFR-MYC cross signaling in murine primary pancreatic ductal epithelial cells

    PubMed Central

    Diersch, Sandra; Wirth, Matthias; Schneeweis, Christian; Jörs, Simone; Geisler, Fabian; Siveke, Jens T.; Rad, Roland; Schmid, Roland M.; Saur, Dieter; Rustgi, Anil K.; Reichert, Maximilian; Schneider, Günter

    2015-01-01

    Epidermal growth factor receptor (EGFR) signaling has a critical role in oncogenic Kras-driven pancreatic carcinogenesis. However, the downstream targets of this signaling network are largely unknown. We developed a novel model system utilizing murine primary pancreatic ductal epithelial cells (PDECs), genetically engineered to allow time-specific expression of oncogenic KrasG12D from the endogenous promoter. We show that primary PDECs are susceptible to KrasG12D-driven transformation and form pancreatic ductal adenocarcinomas (PDAC) in vivo after Cdkn2a inactivation. In addition, we demonstrate that activation of KrasG12D induces an EGFR signaling loop to drive proliferation. Interestingly, pharmacological inhibition of EGFR fails to decrease KrasG12D-activated ERK or PI3K signaling. Instead our data provide novel evidence that EGFR signaling is needed to activate the oncogenic and pro-proliferative transcription factor c-MYC. EGFR and c-MYC have been shown to be essential for pancreatic carcinogenesis. Importantly, our data link both pathways and thereby, explain the crucial role of EGFR for KrasG12D-driven carcinogenesis in the pancreas. PMID:26592448

  3. Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

    PubMed Central

    Gagliano, Nicoletta; Celesti, Giuseppe; Tacchini, Lorenza; Pluchino, Stefano; Sforza, Chiarella; Rasile, Marco; Valerio, Vincenza; Laghi, Luigi; Conte, Vincenzo; Procacci, Patrizia

    2016-01-01

    AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids. PMID:27182158

  4. Inactivation of TGFβ receptor II signalling in pancreatic epithelial cells promotes acinar cell proliferation, acinar-to-ductal metaplasia and fibrosis during pancreatitis.

    PubMed

    Grabliauskaite, Kamile; Saponara, Enrica; Reding, Theresia; Bombardo, Marta; Seleznik, Gitta M; Malagola, Ermanno; Zabel, Anja; Faso, Carmen; Sonda, Sabrina; Graf, Rolf

    2016-02-01

    Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.

  5. Inactivation of TGFβ receptor II signalling in pancreatic epithelial cells promotes acinar cell proliferation, acinar-to-ductal metaplasia and fibrosis during pancreatitis.

    PubMed

    Grabliauskaite, Kamile; Saponara, Enrica; Reding, Theresia; Bombardo, Marta; Seleznik, Gitta M; Malagola, Ermanno; Zabel, Anja; Faso, Carmen; Sonda, Sabrina; Graf, Rolf

    2016-02-01

    Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer. PMID:26510396

  6. The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells

    PubMed Central

    Bosch, Almudena; Panoutsopoulou, Konstantina; Corominas, Josep Maria; Gimeno, Ramón; Moreno-Bueno, Gema; Martín-Caballero, Juan; Morales, Saleta; Lobato, Tania; Martínez-Romero, Carles; Farias, Eduardo F.; Mayol, Xavier; Cano, Amparo; Hernández-Muáoz, Inmaculada

    2014-01-01

    In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy. PMID:24742605

  7. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells.

    PubMed

    Reichert, Maximilian; Takano, Shigetsugu; Heeg, Steffen; Bakir, Basil; Botta, Gregory P; Rustgi, Anil K

    2013-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles duct cells morphologically and, to some extent, at a molecular level. Recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and ductlike cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. This approach is fast (~4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas. PMID:23787893

  8. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells

    PubMed Central

    Reichert, Maximilian; Takano, Shigetsugu; Heeg, Steffen; Bakir, Basil; Botta, Gregory P; Rustgi, Anil K

    2014-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). pDac resembles duct cells morphologically and, to some extent, at a molecular level. recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and duct-like cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. this approach is fast (∼4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas. PMID:23787893

  9. Comparative characterization of stroma cells and ductal epithelium in chronic pancreatitis and pancreatic ductal adenocarcinoma.

    PubMed

    Helm, Ole; Mennrich, Ruben; Petrick, Domantas; Goebel, Lisa; Freitag-Wolf, Sandra; Röder, Christian; Kalthoff, Holger; Röcken, Christoph; Sipos, Bence; Kabelitz, Dieter; Schäfer, Heiner; Oberg, Hans-Heinrich; Wesch, Daniela; Sebens, Susanne

    2014-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive stroma being also present in chronic pancreatitis (CP). Using immunohistochemistry, the stroma of CP and PDAC was comprehensively analyzed and correlated with epithelial/carcinoma-related alterations and clinicopathological patient characteristics. While there were no significant differences between CP and PDAC regarding the distribution of CD3+ T cells and α-SMA+ fibroblasts, proportions of CD4+ and CD8+ T cells were significantly lower and numbers of CD25+(CD4+) and FoxP3+(CD4+) regulatory T cells were greater in PDAC compared with CP. Macrophages were more prevalent in CP, but localized more closely to carcinoma cells in PDAC, as were γδ-T cells. Duct-related FoxP3 and L1CAM expression increased from CP to PDAC, while vimentin expression was similarly abundant in both diseases. Moreover, stromal and epithelial compartments of well-differentiated tumors and CPs shared considerable similarities, while moderately and poorly differentiated tumors significantly differed from CP tissues. Analysis of 27 parameters within each pancreatic disease revealed a significant correlation of i) CD4+ and FoxP3+CD4+ T cells with FoxP3 expression in PDAC cells, ii) α-SMA+ fibroblasts with L1CAM expression and proliferation in PDAC cells, iii) CD3 and CD8 expression with γδ-TCR expression in both pancreatic diseases and iv) CD68+ and CD163+ macrophages with vimentin expression in PDAC cells. High expression of FoxP3, vimentin and L1CAM in PDAC cells as well as a tumor-related localization of macrophages each tended to correlate with higher tumor grade. Multivariate survival analysis revealed a younger age at time of surgery as a positive prognostic marker for PDAC patients with the most frequently operated disease stage T3N1M0. Overall this study identified several interrelationships between stroma and epithelial/carcinoma cells in PDACs but also in CP, which in light of previous experimental data

  10. Proinflammatory Cytokines Induce Endocrine Differentiation in Pancreatic Ductal Cells via STAT3-Dependent NGN3 Activation.

    PubMed

    Valdez, Ivan Achel; Dirice, Ercument; Gupta, Manoj K; Shirakawa, Jun; Teo, Adrian Kee Keong; Kulkarni, Rohit N

    2016-04-19

    A major goal of diabetes research is to develop strategies that replenish pancreatic insulin-producing beta cells. One emerging strategy is to harness pancreatic plasticity-the ability of pancreatic cells to undergo cellular interconversions-a phenomenon implicated in physiological stress and pancreatic injury. Here, we investigate the effects of inflammatory cytokine stress on the differentiation potential of ductal cells in a human cell line, in mouse ductal cells by pancreatic intraductal injection, and during the progression of autoimmune diabetes in the non-obese diabetic (NOD) mouse model. We find that inflammatory cytokine insults stimulate epithelial-to-mesenchymal transition (EMT) as well as the endocrine program in human pancreatic ductal cells via STAT3-dependent NGN3 activation. Furthermore, we show that inflammatory cytokines activate ductal-to-endocrine cell reprogramming in vivo independent of hyperglycemic stress. Together, our findings provide evidence that inflammatory cytokines direct ductal-to-endocrine cell differentiation, with implications for beta cell regeneration. PMID:27068459

  11. Extracellular Ca(2+) sensing in salivary ductal cells.

    PubMed

    Bandyopadhyay, Bidhan C; Swaim, William D; Sarkar, Ankana; Liu, Xibao; Ambudkar, Indu S

    2012-08-31

    Ca(2+) is secreted from the salivary acinar cells as an ionic constituent of primary saliva. Ions such as Na(+) and Cl(-) get reabsorbed whereas primary saliva flows through the salivary ductal system. Although earlier studies have shown that salivary [Ca(2+)] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca(2+) remains enigmatic. Here we report a potential role for the G protein-coupled receptor, calcium-sensing receptor (CSR), in the regulation of Ca(2+) reabsorption by salivary gland ducts. Our data show that CSR is present in the apical region of ductal cells where it is co-localized with transient receptor potential canonical 3 (TRPC3). CSR is activated in isolated salivary gland ducts as well as a ductal cell line (SMIE) by altering extracellular [Ca(2+)] or by aromatic amino acid, L-phenylalanine (L-Phe, endogenous component of saliva), as well as neomycin. CSR activation leads to Ca(2+) influx that, in polarized cells grown on a filter support, is initiated in the luminal region. We show that TRPC3 contributes to Ca(2+) entry triggered by CSR activation. Further, stimulation of CSR in SMIE cells enhances the CSR-TRPC3 association as well as surface expression of TRPC3. Together our findings suggest that CSR could serve as a Ca(2+) sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of [Ca(2+)] from the saliva via TRPC3, thus contributing to maintenance of salivary [Ca(2+)]. CSR could therefore be a potentially important protective mechanism against formation of salivary gland stones (sialolithiasis) and infection (sialoadenitis).

  12. Extracellular Ca2+ Sensing in Salivary Ductal Cells*

    PubMed Central

    Bandyopadhyay, Bidhan C.; Swaim, William D.; Sarkar, Ankana; Liu, Xibao; Ambudkar, Indu S.

    2012-01-01

    Ca2+ is secreted from the salivary acinar cells as an ionic constituent of primary saliva. Ions such as Na+ and Cl− get reabsorbed whereas primary saliva flows through the salivary ductal system. Although earlier studies have shown that salivary [Ca2+] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca2+ remains enigmatic. Here we report a potential role for the G protein-coupled receptor, calcium-sensing receptor (CSR), in the regulation of Ca2+ reabsorption by salivary gland ducts. Our data show that CSR is present in the apical region of ductal cells where it is co-localized with transient receptor potential canonical 3 (TRPC3). CSR is activated in isolated salivary gland ducts as well as a ductal cell line (SMIE) by altering extracellular [Ca2+] or by aromatic amino acid, l-phenylalanine (l-Phe, endogenous component of saliva), as well as neomycin. CSR activation leads to Ca2+ influx that, in polarized cells grown on a filter support, is initiated in the luminal region. We show that TRPC3 contributes to Ca2+ entry triggered by CSR activation. Further, stimulation of CSR in SMIE cells enhances the CSR-TRPC3 association as well as surface expression of TRPC3. Together our findings suggest that CSR could serve as a Ca2+ sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of [Ca2+] from the saliva via TRPC3, thus contributing to maintenance of salivary [Ca2+]. CSR could therefore be a potentially important protective mechanism against formation of salivary gland stones (sialolithiasis) and infection (sialoadenitis). PMID:22778254

  13. Myoepithelial Cell Differentiation Markers in Ductal Carcinoma in Situ Progression

    PubMed Central

    Russell, Tanya D.; Jindal, Sonali; Agunbiade, Samiat; Gao, Dexiang; Troxell, Megan; Borges, Virginia F.; Schedin, Pepper

    2016-01-01

    We describe a preclinical model that investigates progression of early-stage ductal carcinoma in situ (DCIS) and report that compromised myoepithelial cell differentiation occurs before transition to invasive disease. Human breast cancer MCF10DCIS.com cells were delivered into the mouse mammary teat by intraductal injection in the absence of surgical manipulations and accompanying wound-healing confounders. DCIS-like lesions developed throughout the mammary ducts with full representation of human DCIS histologic patterns. Tumor cells were incorporated into the normal mammary epithelium, developed ductal intraepithelial neoplasia and DCIS, and progressed to invasive carcinoma, suggesting the model provides a rigorous approach to study early stages of breast cancer progression. Mammary glands were evaluated for myoepithelium integrity with immunohistochemical assays. Progressive loss of the myoepithelial cell differentiation markers p63, calponin, and α-smooth muscle actin was observed in the mouse myoepithelium surrounding DCIS-involved ducts. p63 loss was an early indicator, calponin loss intermediate, and α-smooth muscle actin a later indicator of compromised myoepithelium. Loss of myoepithelial calponin was specifically associated with gain of the basal marker p63 in adjacent tumor cells. In single time point biopsies obtained from 16 women diagnosed with pure DCIS, a similar loss in myoepithelial cell markers was observed. These results suggest that further research is warranted into the role of myoepithelial cell p63 and calponin expression on DCIS progression to invasive disease. PMID:26343330

  14. Increased breast density correlates with the proliferation-seeking radiotracer (99m)Tc(V)-DMSA uptake in florid epithelial hyperplasia and in mixed ductal carcinoma in situ with invasive ductal carcinoma but not in pure invasive ductal carcinoma or in mild epithelial hyperplasia.

    PubMed

    Papantoniou, Vassilios; Valsamaki, Pipitsa; Sotiropoulou, Evangelia; Tsaroucha, Angeliki; Tsiouris, Spyridon; Sotiropoulou, Maria; Marinopoulos, Spyridon; Kounadi, Evangelia; Karianos, Theodore; Fothiadaki, Athina; Archontaki, Aikaterini; Syrgiannis, Konstantinos; Ptohis, Nikolaos; Makris, Nikolaos; Limouris, Georgios; Antsaklis, Aris

    2011-10-01

    The purpose of this study was to assess the relationship of mammographic breast density (BD) and cell proliferation/focal adhesion kinase activation-seeking radiotracer technetium 99m pentavalent dimercaptosuccinic acid (99mTc(V)-DMSA) uptake in women with different breast histologies, that is, mild epithelial hyperplasia (MEH), florid epithelial hyperplasia (FEH), mixed ductal carcinoma in situ with invasive ductal carcinoma (DCIS + IDC), and pure IDC. Fifty-five women with histologically confirmed mammary pathologies were submitted preoperatively to mammography and 99mTc(V)-DMSA scintimammography. The percentage and intensity of 99mTc(V)-DMSA uptake and the percentage of BD were calculated by computer-assisted methods and compared (t-test) between the breast pathologies. In breasts with increased BD, FEH and DCIS + IDC were found. On the contrary, pure IDC and MEH were identified in breasts with significantly lower BD values. In breasts with increased 99mTc(V)-DMSA area and intensity of uptake, FEH was the main lesion found compared to all other histologies. Linear regression analysis between BD and 99mTc(V)-DMSA uptake area and intensity revealed significant coefficients of correlation (r  =  .689, p < .001 and r  =  .582, p < .001, respectively). Increased BD correlates with the presence of FEH and mixed DCIS + IDC but not with pure IDC or MEH. Its close relationship to 99mTc(V)-DMSA, which also showed an affinity to FEH, indicates that stromal microenvironment may constitute a specific substrate leading to progression to different subtypes of cancerous lesions originating from different pathways.

  15. Stem cells as the root of pancreatic ductal adenocarcinoma

    SciTech Connect

    Balic, Anamaria; Dorado, Jorge; Alonso-Gomez, Mercedes; Heeschen, Christopher

    2012-04-01

    Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.

  16. Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia.

    PubMed

    Pitarresi, Jason R; Liu, Xin; Sharma, Sudarshana M; Cuitiño, Maria C; Kladney, Raleigh D; Mace, Thomas A; Donohue, Sydney; Nayak, Sunayana G; Qu, Chunjing; Lee, James; Woelke, Sarah A; Trela, Stefan; LaPak, Kyle; Yu, Lianbo; McElroy, Joseph; Rosol, Thomas J; Shakya, Reena; Ludwig, Thomas; Lesinski, Gregory B; Fernandez, Soledad A; Konieczny, Stephen F; Leone, Gustavo; Wu, Jinghai; Ostrowski, Michael C

    2016-09-01

    Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin-positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a Kras(G12D)-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2-deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras(G12D) signaling during the initial stages of tumor development. PMID:27659014

  17. Quantification of regenerative potential in primary human mammary epithelial cells

    PubMed Central

    Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  18. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

  19. Characterization of Pancreatic Ductal Cell in Human Islet Preparations

    PubMed Central

    Ichii, Hirohito; Miki, Atsushi; Yamamoto, Toshiyuki; Molano, R. Damaris; Barker, Scott; Mita, Atsuyoshi; Rodriguez-Diaz, Rayner; Klein, Dagmar; Pastori, Ricardo; Alejandro, Rodolfo; Inverardi, Luca; Pileggi, Antonello; Ricordi 6, Camillo

    2013-01-01

    Substantial amounts of non-endocrine cells are implanted as part of human islet grafts, and possible influence of non-endocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for non-endocrine cells due to lack of available methods. Aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDC) for clinical islet transplantation, and to characterize them regarding phenotype, viability and function. We assessed 161 human islet preparations using laser scanning cytometer (LSC/iCys) for phenotypic analysis of non-endocrine cells and flow cytometer (FACS) for PDC viability. PDC and β-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce pro-inflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF), relevant to islet graft outcome. Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDC, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than β-cells (PDC vs. β-cell: 75.5±13.9 and 62.7±18.7 %; p<0.0001). Although β-cells viability was independent from the density, that of PDC was higher as the density from which they were recovered increased. There was no correlation between PDC and β-cells viability (R2=0.0078). PDC sorted from high-density fractions produced significantly higher amount of pro-inflammatory mediators and VEGF, but not TF. PDC isolated from different fractions had different viability and function. The precise characterization and assessment of these cells in addition to β-cells in human islet cell products may be of assistance in understanding their contribution to islet

  20. Ngn3+ endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium

    PubMed Central

    Magenheim, Judith; Klein, Allon M.; Stanger, Ben Z.; Ashery-Padan, Ruth; Sosa-Pineda, Beatriz; Gu, Guoqiang; Dor, Yuval

    2013-01-01

    Summary During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta-cells from stem cells. PMID:21888903

  1. β-cell replacement sources for type 1 diabetes: a focus on pancreatic ductal cells.

    PubMed

    Corritore, Elisa; Lee, Yong-Syu; Sokal, Etienne M; Lysy, Philippe A

    2016-08-01

    Thorough research on the capacity of human islet transplantation to cure type 1 diabetes led to the achievement of 3- to 5-year-long insulin independence in nearly half of transplanted patients. Yet, translation of this technique to clinical routine is limited by organ shortage and the need for long-term immunosuppression, restricting its use to adults with unstable disease. The production of new bona fide β cells in vitro was thus investigated and finally achieved with human pluripotent stem cells (PSCs). Besides ethical concerns about the use of human embryos, studies are now evaluating the possibility of circumventing the spontaneous tumor formation associated with transplantation of PSCs. These issues fueled the search for cell candidates for β-cell engineering with safe profiles for clinical translation. In vivo studies revealed the regeneration capacity of the exocrine pancreas after injury that depends at least partially on facultative progenitors in the ductal compartment. These stimulated subpopulations of pancreatic ductal cells (PDCs) underwent β-cell transdifferentiation through reactivation of embryonic signaling pathways. In vitro models for expansion and differentiation of purified PDCs toward insulin-producing cells were described using cocktails of growth factors, extracellular-matrix proteins and transcription factor overexpression. In this review, we will describe the latest findings in pancreatic β-cell mass regeneration due to adult ductal progenitor cells. We will further describe recent advances in human PDC transdifferentiation to insulin-producing cells with potential for clinical translational studies.

  2. β-cell replacement sources for type 1 diabetes: a focus on pancreatic ductal cells.

    PubMed

    Corritore, Elisa; Lee, Yong-Syu; Sokal, Etienne M; Lysy, Philippe A

    2016-08-01

    Thorough research on the capacity of human islet transplantation to cure type 1 diabetes led to the achievement of 3- to 5-year-long insulin independence in nearly half of transplanted patients. Yet, translation of this technique to clinical routine is limited by organ shortage and the need for long-term immunosuppression, restricting its use to adults with unstable disease. The production of new bona fide β cells in vitro was thus investigated and finally achieved with human pluripotent stem cells (PSCs). Besides ethical concerns about the use of human embryos, studies are now evaluating the possibility of circumventing the spontaneous tumor formation associated with transplantation of PSCs. These issues fueled the search for cell candidates for β-cell engineering with safe profiles for clinical translation. In vivo studies revealed the regeneration capacity of the exocrine pancreas after injury that depends at least partially on facultative progenitors in the ductal compartment. These stimulated subpopulations of pancreatic ductal cells (PDCs) underwent β-cell transdifferentiation through reactivation of embryonic signaling pathways. In vitro models for expansion and differentiation of purified PDCs toward insulin-producing cells were described using cocktails of growth factors, extracellular-matrix proteins and transcription factor overexpression. In this review, we will describe the latest findings in pancreatic β-cell mass regeneration due to adult ductal progenitor cells. We will further describe recent advances in human PDC transdifferentiation to insulin-producing cells with potential for clinical translational studies. PMID:27540464

  3. β-cell replacement sources for type 1 diabetes: a focus on pancreatic ductal cells

    PubMed Central

    Corritore, Elisa; Lee, Yong-Syu; Sokal, Etienne M.; Lysy, Philippe A.

    2016-01-01

    Thorough research on the capacity of human islet transplantation to cure type 1 diabetes led to the achievement of 3- to 5-year-long insulin independence in nearly half of transplanted patients. Yet, translation of this technique to clinical routine is limited by organ shortage and the need for long-term immunosuppression, restricting its use to adults with unstable disease. The production of new bona fide β cells in vitro was thus investigated and finally achieved with human pluripotent stem cells (PSCs). Besides ethical concerns about the use of human embryos, studies are now evaluating the possibility of circumventing the spontaneous tumor formation associated with transplantation of PSCs. These issues fueled the search for cell candidates for β-cell engineering with safe profiles for clinical translation. In vivo studies revealed the regeneration capacity of the exocrine pancreas after injury that depends at least partially on facultative progenitors in the ductal compartment. These stimulated subpopulations of pancreatic ductal cells (PDCs) underwent β-cell transdifferentiation through reactivation of embryonic signaling pathways. In vitro models for expansion and differentiation of purified PDCs toward insulin-producing cells were described using cocktails of growth factors, extracellular-matrix proteins and transcription factor overexpression. In this review, we will describe the latest findings in pancreatic β-cell mass regeneration due to adult ductal progenitor cells. We will further describe recent advances in human PDC transdifferentiation to insulin-producing cells with potential for clinical translational studies. PMID:27540464

  4. Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

    PubMed

    Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-Ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

    2013-11-19

    Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

  5. Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

    PubMed Central

    Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

    2013-01-01

    Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001 PMID:24252877

  6. Analysis of morphological and functional maturation of neoislets generated in vitro from pancreatic ductal cells and their suitability for islet banking and transplantation.

    PubMed

    Katdare, M R; Bhonde, R R; Parab, P B

    2004-07-01

    The pancreatic ductal stem cells are known to differentiate into islets of Langerhans; however, their yield is limited and the islet population is not defined. Therefore, the aims of the present study were to improvise a methodology for obtaining large numbers of islets in vitro and to characterize their morphological and functional status for islet cell banking and transplantation. Pancreatic ductal epithelial cell cultures were set in serum-free medium. Monolayers of epithelial cells in culture gave rise to islet-like clusters within 3-4 weeks. The identity of neoislets was confirmed by dithizone staining and analysis of the gene expression for endocrine markers by reverse transcriptase-polymerase chain reaction (RT-PCR). The islet population obtained was analysed by image analysis and insulin secretion in response to secretagogues. The cellular extracts from neoislets were immunoreactive to anti-insulin antibody and expressed insulin, glucagon, GLUT-2, PDX-1 and Reg-1 genes. The islets generated within 3-4 weeks exhibited a mixed population of large- and small-sized islets with clear cut dichotomy in the pattern of their insulin secretion in response to L-arginine and glucose. These neoislets maintained their structural and functional integrity on cryopreservation and transplantation indicating their suitability for islet cell banking. Thus, the present study describes an improved method for obtaining a constant supply of large numbers of islets from pancreatic ductal stem cell cultures. The newly generated islets undergo functional maturation indicating their suitability for transplantation.

  7. Pancreatic ductal adenocarcinoma cell lines display a plastic ability to bi‑directionally convert into cancer stem cells.

    PubMed

    Dalla Pozza, Elisa; Dando, Ilaria; Biondani, Giulia; Brandi, Jessica; Costanzo, Chiara; Zoratti, Elisa; Fassan, Matteo; Boschi, Federico; Melisi, Davide; Cecconi, Daniela; Scupoli, Maria Teresa; Scarpa, Aldo; Palmieri, Marta

    2015-03-01

    Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when metastatic events have occurred. Cancer stem cells (CSCs) play an important role in tumor initiation, metastasis, chemoresistance and relapse. A growing number of studies have suggested that CSCs exist in a dynamic equilibrium with more differentiated cancer cells via a bi‑directional regeneration that is dependent on the environmental stimuli. In this investigation, we obtain, by using a selective medium, PDAC CSCs from five out of nine PDAC cell lines, endowed with different tumorsphere‑forming ability. PDAC CSCs were generally more resistant to the action of five anticancer drugs than parental cell lines and were characterized by an increased expression of EpCAM and CD44v6, typical stem cell surface markers, and a decreased expression of E‑cadherin, the main marker of the epithelial state. PDAC CSCs were able to re‑differentiate into parental cells once cultured in parental growth condition, as demonstrated by re‑acquisition of the epithelial morphology, the decreased expression levels of EpCAM and CD44v6 and the increased sensitivity to anticancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumor mass and showed a higher metastatic activity compared to parental cells. The present study demonstrates the ability to obtain CSCs from several PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability renders them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarkers and to screen new therapeutic compounds.

  8. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  9. Retinal pigment epithelial cell proliferation

    PubMed Central

    Temple, Sally

    2015-01-01

    The human retinal pigment epithelium forms early in development and subsequently remains dormant, undergoing minimal proliferation throughout normal life. Retinal pigment epithelium proliferation, however, can be activated in disease states or by removing retinal pigment epithelial cells into culture. We review the conditions that control retinal pigment epithelial proliferation in culture, in animal models and in human disease and interpret retinal pigment epithelium proliferation in context of the recently discovered retinal pigment epithelium stem cell that is responsible for most in vitro retinal pigment epithelial proliferation. Retinal pigment epithelial proliferation-mediated wound repair that occurs in selected macular diseases is contrasted with retinal pigment epithelial proliferation-mediated fibroblastic scar formation that underlies proliferative vitreoretinopathy. We discuss the role of retinal pigment epithelial proliferation in age-related macular degeneration which is reparative in some cases and destructive in others. Macular retinal pigment epithelium wound repair and regression of choroidal neovascularization are more pronounced in younger than older patients. We discuss the possibility that the limited retinal pigment epithelial proliferation and latent wound repair in older age-related macular degeneration patients can be stimulated to promote disease regression in age-related macular degeneration. PMID:26041390

  10. PD2/Paf1 depletion in pancreatic acinar cells promotes acinar-to-ductal metaplasia

    PubMed Central

    Dey, Parama; Rachagani, Satyanarayana; Vaz, Arokia P.; Ponnusamy, Moorthy P.; Batra, Surinder K.

    2014-01-01

    Pancreatic differentiation 2 (PD2), a PAF (RNA Polymerase II Associated Factor) complex subunit, is overexpressed in pancreatic cancer cells and has demonstrated potential oncogenic property. Here, we report that PD2/Paf1 expression was restricted to acinar cells in the normal murine pancreas, but its expression increased in the ductal cells of Pdx1Cre; KrasG12D (KC) mouse model of pancreatic cancer with increasing age, showing highest expression in neoplastic ductal cells of 50 weeks old mice. PD2/Paf1 was specifically expressed in amylase and CK19 double positive metaplastic ducts, representing intermediate structures during pancreatic acinar-to-ductal metaplasia (ADM). Similar PD2/Paf1 expression was observed in murine pancreas that exhibited ADM-like histology upon cerulein challenge. In normal mice, cerulein-mediated inflammation induced a decrease in PD2/Paf1 expression, which was later restored upon recovery of the pancreatic parenchyma. In KC mice, however, PD2/Paf1 mRNA level continued to decrease with progressive dysplasia and subsequent neoplastic transformation. Additionally, knockdown of PD2/Paf1 in pancreatic acinar cells resulted in the abrogation of Amylase, Elastase and Lipase (acinar marker) mRNA levels with simultaneous increase in CK19 and CAII (ductal marker) transcripts. In conclusion, our studies indicate loss of PD2/Paf1 expression during acinar transdifferentiation in pancreatic cancer initiation and PD2/Paf1 mediated regulation of lineage specific markers. PMID:24947474

  11. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  12. Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

    PubMed Central

    Iglesias, Juan Manuel; Cairney, Claire J.; Ferrier, Roderick K.; McDonald, Laura; Soady, Kelly; Kendrick, Howard; Pringle, Marie-Anne; Morgan, Reginald O.; Martin, Finian; Smalley, Matthew J.; Blyth, Karen; Stein, Torsten

    2015-01-01

    We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin. PMID:25803307

  13. Detection of circulating pancreas epithelial cells in patients with pancreatic cystic lesions.

    PubMed

    Rhim, Andrew D; Thege, Fredrik I; Santana, Steven M; Lannin, Timothy B; Saha, Trisha N; Tsai, Shannon; Maggs, Lara R; Kochman, Michael L; Ginsberg, Gregory G; Lieb, John G; Chandrasekhara, Vinay; Drebin, Jeffrey A; Ahmad, Nuzhat; Yang, Yu-Xiao; Kirby, Brian J; Stanger, Ben Z

    2014-03-01

    Hematogenous dissemination is thought to be a late event in cancer progression. We recently showed in a genetic model of pancreatic ductal adenocarcinoma that pancreas cells can be detected in the bloodstream before tumor formation. To confirm these findings in humans, we used microfluidic geometrically enhanced differential immunocapture to detect circulating pancreas epithelial cells in patient blood samples. We captured more than 3 circulating pancreas epithelial cells/mL in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with pancreatic ductal adenocarcinoma, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors are detected, which might be used in risk assessment. PMID:24333829

  14. Detection of circulating pancreas epithelial cells in patients with pancreatic cystic lesions.

    PubMed

    Rhim, Andrew D; Thege, Fredrik I; Santana, Steven M; Lannin, Timothy B; Saha, Trisha N; Tsai, Shannon; Maggs, Lara R; Kochman, Michael L; Ginsberg, Gregory G; Lieb, John G; Chandrasekhara, Vinay; Drebin, Jeffrey A; Ahmad, Nuzhat; Yang, Yu-Xiao; Kirby, Brian J; Stanger, Ben Z

    2014-03-01

    Hematogenous dissemination is thought to be a late event in cancer progression. We recently showed in a genetic model of pancreatic ductal adenocarcinoma that pancreas cells can be detected in the bloodstream before tumor formation. To confirm these findings in humans, we used microfluidic geometrically enhanced differential immunocapture to detect circulating pancreas epithelial cells in patient blood samples. We captured more than 3 circulating pancreas epithelial cells/mL in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with pancreatic ductal adenocarcinoma, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors are detected, which might be used in risk assessment.

  15. Selective functionalization of nanofiber scaffolds to regulate salivary gland epithelial cell proliferation and polarity.

    PubMed

    Cantara, Shraddha I; Soscia, David A; Sequeira, Sharon J; Jean-Gilles, Riffard P; Castracane, James; Larsen, Melinda

    2012-11-01

    Epithelial cell types typically lose apicobasal polarity when cultured on 2D substrates, but apicobasal polarity is required for directional secretion by secretory cells, such as salivary gland acinar cells. We cultured salivary gland epithelial cells on poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds that mimic the basement membrane, a specialized extracellular matrix, and examined cell proliferation and apicobasal polarization. Although cells proliferated on nanofibers, chitosan-coated nanofiber scaffolds stimulated proliferation of salivary gland epithelial cells. Although apicobasal cell polarity was promoted by the nanofiber scaffolds relative to flat surfaces, as determined by the apical localization of ZO-1, it was antagonized by the presence of chitosan. Neither salivary gland acinar nor ductal cells fully polarized on the nanofiber scaffolds, as determined by the homogenous membrane distribution of the mature tight junction marker, occludin. However, nanofiber scaffolds chemically functionalized with the basement membrane protein, laminin-111, promoted more mature tight junctions, as determined by apical localization of occludin, but did not affect cell proliferation. To emulate the multifunctional capabilities of the basement membrane, bifunctional PLGA nanofibers were generated. Both acinar and ductal cell lines responded to signals provided by bifunctional scaffolds coupled to chitosan and laminin-111, demonstrating the applicability of such scaffolds for epithelial cell types.

  16. Preadipocyte factor 1 induces pancreatic ductal cell differentiation into insulin-producing cells.

    PubMed

    Rhee, Marie; Lee, Seung-Hwan; Kim, Ji-Won; Ham, Dong-Sik; Park, Heon-Seok; Yang, Hae Kyung; Shin, Ju-Young; Cho, Jae-Hyoung; Kim, Young-Bum; Youn, Byung-Soo; Sul, Hei Sook; Yoon, Kun-Ho

    2016-01-01

    The preadipocyte factor 1 (Pref-1) is involved in the proliferation and differentiation of various precursor cells. However, the intracellular signaling pathways that control these processes and the role of Pref-1 in the pancreas remain poorly understood. Here, we showed that Pref-1 induces insulin synthesis and secretion via two independent pathways. The overexpression of Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translocation of FOXO1 and PDX1 and led to the differentiation of human pancreatic ductal cells into β-like cells and an increase in insulin synthesis. Concurrently, Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics analysis identified the Rab43 GTPase-activating protein as a downstream target of Akt. A serial activation of both proteins induced various granular protein syntheses which led to enhanced glucose-stimulated insulin secretion. In a pancreatectomised diabetic animal model, exogenous Pref-1 improved glucose homeostasis by accelerating pancreatic ductal and β-cell regeneration after injury. These data establish a novel role for Pref-1, opening the possibility of applying this molecule to the treatment of diabetes. PMID:27044861

  17. Implications of the Notch1-Snail/Slug-epithelial to mesenchymal transition axis for lymph node metastasis in infiltrating ductal carcinoma.

    PubMed

    Cao, Yu-Wen; Wan, Guo-Xing; Sun, Jian-Ping; Cui, Xiao-Bin; Hu, Jian-Ming; Liang, Wei-Hua; Zheng, Yu-Qin; Li, Wen-Qin; Li, Feng

    2015-02-01

    Emerging evidence suggests that activation of the Notch1 signaling pathway inducing epithelial to mesenchymal transition (EMT) mediated by Snail/Slug promotes invasion and metastasis of breast cancer cells in vitro. However, the implication of the Notch1-Snail/Slug-EMT axis in breast cancer patients remains unclear. A total of 200 formalin-fixed paraffin-embedded samples of invasive ductal carcinoma (IDC), and 37 adjacent non-neoplastic tissue (ANNT) samples from patients who had not been treated with neoadjuvant therapy were examined. Expression of Notch1, Slug, Snail, E-cadherin, N-cadherin, and vimentin was determined by immunohistochemistry on tissue microarrays (TMAs). The correlation between protein expression and clinicopathological characteristics of breast cancer patients was also evaluated. Results showed that a significantly high percentage of cases with high expression of Notch1 (74%, 148/200), Slug (36%, 72/200), Snail (62%, 124/200), and N-cadherin (77%, 153/200) and a low percentage of cases with high expression of E-cadherin (27%, 54/200) were observed in IDC compared to those in ANNTs. High Notch1, Slug, Snail, and N-cadherin expression and low E-cadherin expression in patients with IDC were significantly correlated with lymph node metastasis. In addition, correlation analysis results revealed that high Notch1 expression was significantly associated with high Slug, Snail, and N-cadherin expression and low E-cadherin expression in IDC. Furthermore, a high Snail expression was significantly associated with low E-cadherin expression, and a high Slug expression was found to be significantly associated with increased N-cadherin expression in patients with IDC. Hence, our study suggested that the Notch1-Snail/Slug-EMT axis may be implicated in the lymph node metastasis affecting patients with IDC. PMID:25645984

  18. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  19. Current management of ductal carcinoma in situ.

    PubMed

    Barth, A; Brenner, R J; Giuliano, A E

    1995-10-01

    Ductal carcinoma in situ represents a biologically and histologically heterogeneous group of lesions characterized by the proliferation of neoplastic epithelial cells confined to the ducts of the breast. Before screening mammography, ductal carcinoma in situ was considered uncommon; patients were usually diagnosed by a breast mass or bloody nipple discharge, and their treatment was mastectomy. Today it represents 20% to 30% of mammographically detected breast cancers and 10% to 15% of all diagnosed breast cancers in the United States. The invariable progression of this cancer to invasive breast cancer requiring mastectomy has been challenged, but because most patients have been treated with mastectomy, knowledge about ductal carcinoma in situ is limited and primarily based on retrospective data. Further insight will emerge from randomized prospective studies that are near completion. Currently available data indicate that breast-conserving treatments are valid alternatives to mastectomy for most patients with this disease. PMID:7483593

  20. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

    PubMed Central

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  1. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells.

    PubMed

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na(+) channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  2. Secretory component: a glandular epithelial cell marker.

    PubMed Central

    Harris, J. P.; South, M. A.

    1981-01-01

    Secretory component (SC) has been demonstrated to be produced by both normal and malignantly transformed glandular epithelial cells. By an indirect immunofluorescent technique, this study surveys tumors of varied cellular origin in order to determine the reliability of SC as a marker for tumor cells derived from glandular epithelium. Both primary and metastatic tumors of glandular epithelial origin demonstrated SC fluorescence, while nonglandular epithelial tumors did not. This observation was extended to live single-cell preparations, which demonstrated intense cell-surface fluorescence only when glandular epithelial tumors cells were examined. Additionally, fixed, cytocentrifuged, single-cell preparations of glandular epithelial tumors demonstrated cytoplasmic SC fluorescence. When breast carcinoma was examined, all cases demonstrated SC, regardless of the degree of differentiation. This assay appears to have useful clinical application in that the finding of SC provides indication of the glandular epithelial origin of a malignantly transformed cell. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6271014

  3. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

    Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid. PMID:196249

  4. Resistance of cyclooxygenase-2 expressing pancreatic ductal adenocarcinoma cells against γδ T cell cytotoxicity

    PubMed Central

    Gonnermann, Daniel; Oberg, Hans-Heinrich; Kellner, Christian; Peipp, Matthias; Sebens, Susanne; Kabelitz, Dieter; Wesch, Daniela

    2015-01-01

    The prostaglandin (PG) synthetase cyclooxygenase 2 (Cox-2) promotes tumorigenesis, tumor progression, and metastasis in a variety of human cancer entities including pancreatic ductal adenocarcinoma (PDAC). In this study, we demonstrate that in PDAC cells such as Colo357 cells, enhanced Cox-2 expression and increased release of the Cox-2 metabolite prostaglandin E2 (PGE2) promotes resistance against γδ T cell-mediated lysis. Co-culture with activated γδ T cells induced an upregulation of Cox-2 expression in Colo357 cells, and thereby an enhanced PGE2 release, in response to tumor necrosis factor α (TNFα) secretion from γδ T cells. The PGE2-mediated inhibition of γδ T cell cytotoxicity against Cox-2-expressing PDAC cells can be partially overcome by Cox-2 inhibitors. Our results show that differences between PDAC cells in regards to sensitivity to γδ T-cell cytotoxicity can be due to distinct levels of Cox-2 expression associated with varying amounts of PGE2 release. While γδ T cell cytotoxicity against PDAC cells expressing low levels of Cox-2 can be effectively enhanced by tribody [(Her2)2×Vγ9] with specificity for Vγ9 T cell receptor and HER-2/neu on PDAC cells, a combination of tribody [(Her2)2×Vγ9] and Cox-2 inhibitor is necessary to induce complete lysis of Cox-2 high expressing Colo357. In conclusion, our results suggest that the application of tribody [(Her2)2×Vγ9] that enhances γδ T-cell cytotoxicity and Cox-2 inhibitors that overcome PGE2-mediated resistance of PDAC cells to the cytotoxic activity of γδ T cells might offer a promising combined immunotherapy for pancreatic cancer. PMID:25949900

  5. Uptake and metabolism of D-glucose in isolated acinar and ductal cells from rat submandibular glands.

    PubMed

    Cetik, Sibel; Rzajeva, Aigun; Hupkens, Emeline; Malaisse, Willy J; Sener, Abdullah

    2014-07-01

    The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of d-glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of d-[U-(14) C]glucose and its non-metabolised analogue 3-O-[(14) C-methyl]-d-glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of d-glucose and 3-O-methyl-d-glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of d-glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired d-glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca(2+) or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin-sensitive, cytochalasin B-sensitive and phloridzin-sensitive transport of d-glucose across the plasma membrane.

  6. Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines

    PubMed Central

    Pizzuto, Matteo S.; Silic-Benussi, Micol; Pavone, Silvia; Ciminale, Vincenzo; Capua, Ilaria

    2014-01-01

    ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas. IMPORTANCE Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These

  7. Oleic acid and glucose regulate glucagon-like peptide 1 receptor expression in a rat pancreatic ductal cell line

    SciTech Connect

    Zhang, Leshuai W.; McMahon Tobin, Grainne A.; Rouse, Rodney L.

    2012-10-15

    The glucagon-like peptide 1 receptor (GLP1R) plays a critical role in glucose metabolism and has become an important target for a growing class of drugs designed to treat type 2 diabetes. In vitro studies were designed to investigate the effect of the GLP1R agonist, exenatide (Ex4), in “on-target” RIN-5mF (islet) cells as well as in “off-target” AR42J (acinar) and DSL-6A/C1 (ductal) cells in a diabetic environment. Ex4 increased islet cell proliferation but did not affect acinar cells or ductal cells at relevant concentrations. A high caloric, high fat diet is a risk factor for impaired glucose tolerance and type-2 diabetes. An in vitro Oleic acid (OA) model was used to investigate the effect of Ex4 in a high calorie, high fat environment. At 0.1 and 0.4 mM, OA mildly decreased the proliferation of all pancreatic cell types. Ex4 did not potentiate the inhibitory effect of OA on cell proliferation. Akt phosphorylation in response to Ex4 was diminished in OA-treated ductal cells. GLP1R protein detected by western blot was time and concentration dependently decreased after glucose stimulation in OA-treated ductal cells. In ductal cells, OA treatment altered the intracellular localization of GLP1R and its co-localization with early endosome and recycling endosomes. Chloroquine (lysosomal inhibitor), N-acetyl-L-cysteine (reactive oxygen species scavenger) and wortmannin (a phosphatidylinositol-3-kinase inhibitor), fully or partially, rescued GLP1R protein in OA-pretreated, glucose-stimulated ductal cells. The impact of altered regulation on phenotype/function is presently unknown. However, these data suggest that GLP1R regulation in ductal cells can be altered by a high fat, high calorie environment. -- Highlights: ► Exenatide did not inhibit islet, acinar or ductal cell proliferation. ► GLP1R protein decreased after glucose stimulation in oleic acid-treated ductal cells. ► Oleic acid treatment altered localization of GLP1R with early and recycling

  8. TGF-β1 promotes acinar to ductal metaplasia of human pancreatic acinar cells

    PubMed Central

    Liu, Jun; Akanuma, Naoki; Liu, Chengyang; Naji, Ali; Halff, Glenn A.; Washburn, William K.; Sun, Luzhe; Wang, Pei

    2016-01-01

    Animal studies suggest that pancreatitis-induced acinar-to-ductal metaplasia (ADM) is a key event for pancreatic ductal adenocarcinoma (PDAC) initiation. However, there has not been an adequate system to explore the mechanisms of human ADM induction. We have developed a flow cytometry-based, high resolution lineage tracing method and 3D culture system to analyse ADM in human cells. In this system, well-known mouse ADM inducers did not promote ADM in human cells. In contrast, TGF-β1 efficiently converted human acinar cells to duct-like cells (AD) in a SMAD-dependent manner, highlighting fundamental differences between the species. Functionally, AD cells gained transient proliferative capacity. Furthermore, oncogenic KRAS did not induce acinar cell proliferation, but did sustain the proliferation of AD cells, suggesting that oncogenic KRAS requires ADM-associated-changes to promote PDAC initiation. This ADM model provides a novel platform to explore the mechanisms involved in the development of human pancreatic diseases. PMID:27485764

  9. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

    SciTech Connect

    Montesano, Roberto Sarkoezi, Rita; Schramek, Herbert

    2008-09-12

    Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

  10. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.

  11. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation. PMID:27520504

  12. Basal cytokeratin phenotypes of myoepithelial cells indicates the origin of ductal carcinomas in situ of the breast.

    PubMed

    Chen, Ling; Yin, Xiaona; Lu, Shanshan; Chen, Guorong; Dong, Lei

    2015-09-01

    Terminal duct lobular unit (TDLU) is widely accepted as the origin of ductal carcinoma in situ of breast. The differentiation states of myoepithelial cells of breast ductal system hint the development of breast hyperplastic lesions. Basal cytokeratin (CK) phenotypes indicate the differentiation of myoepithelial cells. Using antibodies of CK5/6, CK14, and CK17, this study reports the basal CK phenotypes of myoepithelial cells in 20 foci of normal breast, 20 usual ductal hyperplasias, 36 ductal carcinomas in situ (DCIS), and 28 sclerosing adenosis (SA). The results showed that the positive staining of basal CKs of myoepithelial cells in normal ducts were significantly higher than those in normal lobules. The basal CK expression of myoepithelial cells of DCIS and usual ductal hyperplasia was similar to that of normal duct, whereas that of SA was similar to that of normal lobule. We propose a modified model of TDLU origin of intraductal carcinoma that most of DCIS originate from terminal ducts of TDLU, whereas most SA originate from lobules.

  13. Clara epithelial cell depletion in the lung.

    PubMed

    Sonar, Sanchaita S; Dudda, Jan C

    2013-01-01

    The bronchial epithelium has been increasingly recognized as an important immunomodulatory compartment in asthma and other lung diseases. Clara cells, which comprise the nonciliated secretory epithelial cells, are an important epithelial cell type with functions in the regulation of lung homeostasis and inflammation. Using naphthalene, Clara cells can be depleted within 24 h and regenerate by 1 month, hence, providing an easy method to study the impact of Clara cells on lung inflammation.

  14. Differential expression of epithelial cell adhesion molecule in salivary gland neoplasms.

    PubMed

    Phattarataratip, Ekarat; Masorn, Marisa; Jarupoonphol, Werapong; Supatthanayut, Sirinpaporn; Saeoweiang, Pichanee

    2016-10-01

    Epithelial cell adhesion molecule (EpCAM) is the epithelial-specific molecule expressed on various epithelial cell types. The function of EpCAM involves cellular adhesion, proliferation, and signaling in both normal tissues and cancers. The purposes of this study were to investigate the EpCAM expression in salivary gland neoplasms and examine its relationship with pathologic characteristics. Forty-two cases of salivary gland neoplasms, including 20 mucoepidermoid carcinomas (MECs), 11 adenoid cystic carcinomas (ACCs), 9 pleomorphic adenomas (PAs), and 2 polymorphous low-grade adenocarcinomas (PLGAs) were enrolled. Epithelial cell adhesion molecule expression was analyzed immunohistochemically using MOC-31 and BerEP4 antibodies. Results showed that the majority of MECs and all PLGAs showed EpCAM expression in more than 50% of neoplastic cells, whereas most PAs and ACCs did not express this protein. In MECs, most EpCAM-positive neoplastic cells were clear cells, glandular epithelial cells, and intermediate cells, whereas squamous cells and mucous cells were largely negative. The expression was limited to ductal epithelium in EpCAM-positive PAs and ACCs. The decreased EpCAM expression in MECs was significantly associated with microscopically diminished cystic components, the presence of small nest invasion at invasive front, cellular anaplasia, vascular invasion, and high pathologic grade. These data suggested that EpCAM showed different expression pattern among salivary gland neoplasms and in different grades of MECs. PMID:27649957

  15. Human glomerular epithelial cell proteoglycans

    SciTech Connect

    Thomas, G.J.; Jenner, L.; Mason, R.M.; Davies, M. )

    1990-04-01

    Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. Heparan sulfate proteoglycan I (HSPG-I; Kav 6B 0.04) was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPG-II (Kav 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; Kav 6B 0.4-0.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecular-mass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -II released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (Kav 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (Kav 6B 0.25-0.6) is present in the cell extract and like HS-GAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -II and CSPG-I and -II are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate.

  16. Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells.

    PubMed

    Pochet, Stéphanie; Métioui, Mourad; Grosfils, Katrina; Gómez-Muñoz, Antonio; Marino, Aida; Dehaye, Jean-Paul

    2003-01-01

    The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF(4)(-), two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55-60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC-PKC-tyrosine kinase pathway. PMID:12401525

  17. CD44 expression in intraoral salivary ductal papillomas and oral papillary squamous cell carcinoma.

    PubMed

    Fitzpatrick, Sarah G; Montague, Lindsay J; Cohen, Donald M; Bhattacharyya, Indraneel

    2013-06-01

    CD44 is a transmembrane adhesion molecule which has been previously shown to be useful in the differentiation of benign papillary lesions from invasive carcinoma in several different areas including sinonasal mucosa and breast tissue. CD44 expression has previously been shown to be lost in invasive carcinoma and retained in benign papillary lesions in both of the above locations. In addition, studies have evaluated oral mucosal lesions for CD44 expression and found a loss with invasive squamous cell carcinoma when compared to normal epithelium, hyperplasia, and squamous papillomas, which stained particularly strongly. To the best of our knowledge, no study has evaluated CD44 expression when comparing salivary ductal papillomas in comparison to oral papillary SCCA. In this study 18 cases of intraductal papilloma were compared to 19 cases of oral papillary SCCA. Within the ductal papilloma group, all cases stained either absent (6%), weakly (33%), or moderately (61%) with 76% expressing the stain diffusely and 24% focally. In comparison, the papillary squamous cell carcinoma cases expressed the CD44 moderately (26%) or strongly (74%) with 100 % showing diffuse staining. Thus, the CD44 expression was contrary to expectation based on previous studies, which we hypothesize is due to the extremely well differentiated nature of papillary SCCA which expressed CD44 staining compatible with levels previously reported with oral squamous papillomas than invasive carcinoma.

  18. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  19. Role of HGF in epithelial–stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ

    PubMed Central

    2013-01-01

    Introduction Basal-like and luminal breast cancers have distinct stromal–epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal–epithelial interactions evolve in benign and pre-invasive lesions. Methods To study epithelial–stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis. Results Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basal-like subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival. Conclusions Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that

  20. Murine amniotic fluid stem cells contribute mesenchymal but not epithelial components to reconstituted mammary ducts

    PubMed Central

    2010-01-01

    Introduction Amniotic fluid harbors cells indicative of all three germ layers, and pluripotent fetal amniotic fluid stem cells (AFSs) are considered potentially valuable for applications in cellular therapy and tissue engineering. We investigated whether it is possible to direct the cell fate of AFSs in vivo by transplantation experiments into a particular microenvironment, the mammary fat pad. This microenvironment provides the prerequisites to study stem cell function and the communication between mesenchymal and epithelial cells. On clearance of the endogenous epithelium, the ductal tree can be reconstituted by the transfer of exogenously provided mammary stem cells. Analogously, exogenously provided stem cells from other tissues can be investigated for their potential to contribute to mammary gland regeneration. Methods We derived pluripotent murine AFSs, measured the expression of stem cell markers, and confirmed their in vitro differentiation potential. AFSs were transplanted into cleared and non cleared fat pads of immunocompromised mice to evaluate their ability to assume particular cell fates under the instructive conditions of the fat-pad microenvironment and the hormonal stimulation during pregnancy. Results Transplantation of AFSs into cleared fat pads alone or in the presence of exogenous mammary epithelial cells caused their differentiation into stroma and adipocytes and replaced endogenous mesenchymal components surrounding the ducts in co-transplantation experiments. Similarly, transplantation of AFSs into fat pads that had not been previously cleared led to AFS-derived stromal cells surrounding the elongating endogenous ducts. AFSs expressed the marker protein α-SMA, but did not integrate into the myoepithelial cell layer of the ducts in virgin mice. With pregnancy, a small number of AFS-derived cells were present in acinar structures. Conclusions Our data demonstrate that the microenvironmental cues of the mammary fat pad cause AFSs to

  1. An Increased Abundance of Tumor-Infiltrating Regulatory T Cells Is Correlated with the Progression and Prognosis of Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Tang, Yichen; Xu, Xuejun; Guo, Shixiang; Zhang, Chaobin; Tang, Yan; Tian, Yi; Ni, Bing; Lu, Binfeng; Wang, Huaizhi

    2014-01-01

    CD4+CD25+Foxp3+ regulatory T cells (Tregs) can inhibit cytotoxic responses. Though several studies have analyzed Treg frequency in the peripheral blood mononuclear cells (PBMCs) of pancreatic ductal adenocarcinoma (PDA) patients using flow cytometry (FCM), few studies have examined how intratumoral Tregs might contribute to immunosuppression in the tumor microenvironment. Thus, the potential role of intratumoral Tregs in PDA patients remains to be elucidated. In this study, we found that the percentages of Tregs, CD4+ T cells and CD8+ T cells were all increased significantly in tumor tissue compared to control pancreatic tissue, as assessed via FCM, whereas the percentages of these cell types in PBMCs did not differ between PDA patients and healthy volunteers. The percentages of CD8+ T cells in tumors were significantly lower than in PDA patient PBMCs. In addition, the relative numbers of CD4+CD25+Foxp3+ Tregs and CD8+ T cells were negatively correlated in the tissue of PDA patients, and the abundance of Tregs was significantly correlated with tumor differentiation. Additionally, Foxp3+ T cells were observed more frequently in juxtatumoral stroma (immediately adjacent to the tumor epithelial cells). Patients showing an increased prevalence of Foxp3+ T cells had a poorer prognosis, which was an independent factor for patient survival. These results suggest that Tregs may promote PDA progression by inhibiting the antitumor immunity of CD8+ T cells at local intratumoral sites. Moreover, a high proportion of Tregs in tumor tissues may reflect suppressed antitumor immunity. PMID:24637664

  2. Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

    PubMed Central

    Tsunoda, Kazuyuki; Nakagawa, Taneaki; Tsubota, Kazuo

    2016-01-01

    Purpose Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). Methods SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. Results SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. Conclusions Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium. PMID:26800086

  3. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    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. The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation.

    PubMed

    Wright, Bernice; Hopkinson, Andrew; Leyland, Martin; Connon, Che J

    2013-01-01

    Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P≤0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.

  5. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  6. A case of renal cell carcinoma metastasizing to invasive ductal breast carcinoma.

    PubMed

    Chen, Tai-Di; Lee, Li-Yu

    2014-02-01

    Tumor-to-tumor metastasis is an uncommon but well-documented phenomenon. We present a case of a clear cell renal cell carcinoma (RCC) metastasizing to an invasive ductal carcinoma (IDC) of the breast. A 74-year-old woman with a past history of clear cell RCC status after radical nephrectomy underwent right modified radical mastectomy for an enlarging breast mass 3 years after nephrectomy. Histological examination revealed a small focus with distinct morphological features similar to clear cell RCC encased in the otherwise typical IDC. Immunohistochemical studies showed that this focus was positive for CD10 and vimentin, in contrast to the surrounding IDC, which was negative for both markers and positive for Her2/neu. Based on the histological and immunohistochemical features, the patient was diagnosed with metastasis of clear cell RCC to the breast IDC. To the best of our knowledge, this is the first reported case of a breast neoplasm as the recipient tumor in tumor-to-tumor metastasis. PMID:24530247

  7. Role of YAP and TAZ in pancreatic ductal adenocarcinoma and in stellate cells associated with cancer and chronic pancreatitis.

    PubMed

    Morvaridi, Susan; Dhall, Deepti; Greene, Mark I; Pandol, Stephen J; Wang, Qiang

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic and inflammatory microenvironment that is formed primarily by activated, myofibroblast-like, stellate cells. Although the stellate cells are thought to contribute to tumorigenesis, metastasis and drug resistance of PDAC, the signaling events involved in activation of the stellate cells are not well defined. Functioning as transcription co-factors, Yes-associated protein (YAP) and its homolog transcriptional co-activator with PDZ-binding motif (TAZ) modulate the expression of genes involved in various aspects of cellular functions, such as proliferation and mobility. Using human tissues we show that YAP and TAZ expression is restricted to the centroacinar and ductal cells of normal pancreas, but is elevated in cancer cells. In particular, YAP and TAZ are expressed at high levels in the activated stellate cells of both chronic pancreatitis and PDAC patients as well as in the islets of Langerhans in chronic pancreatitis tissues. Of note, YAP is up regulated in both acinar and ductal cells following induction of acute and chronic pancreatitis in mice. These findings indicate that YAP and TAZ may play a critical role in modulating pancreatic tissue regeneration, neoplastic transformation, and stellate cell functions in both PDAC and pancreatitis. PMID:26567630

  8. Role of YAP and TAZ in pancreatic ductal adenocarcinoma and in stellate cells associated with cancer and chronic pancreatitis

    PubMed Central

    Morvaridi, Susan; Dhall, Deepti; Greene, Mark I.; Pandol, Stephen J.; Wang, Qiang

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic and inflammatory microenvironment that is formed primarily by activated, myofibroblast-like, stellate cells. Although the stellate cells are thought to contribute to tumorigenesis, metastasis and drug resistance of PDAC, the signaling events involved in activation of the stellate cells are not well defined. Functioning as transcription co-factors, Yes-associated protein (YAP) and its homolog transcriptional co-activator with PDZ-binding motif (TAZ) modulate the expression of genes involved in various aspects of cellular functions, such as proliferation and mobility. Using human tissues we show that YAP and TAZ expression is restricted to the centroacinar and ductal cells of normal pancreas, but is elevated in cancer cells. In particular, YAP and TAZ are expressed at high levels in the activated stellate cells of both chronic pancreatitis and PDAC patients as well as in the islets of Langerhans in chronic pancreatitis tissues. Of note, YAP is up regulated in both acinar and ductal cells following induction of acute and chronic pancreatitis in mice. These findings indicate that YAP and TAZ may play a critical role in modulating pancreatic tissue regeneration, neoplastic transformation, and stellate cell functions in both PDAC and pancreatitis. PMID:26567630

  9. Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland.

    PubMed

    Matsuura, Sachiko; Koyama, Noriko; Kashimata, Masanori; Hayashi, Haruki; Kikuta, Akio

    2007-09-01

    Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland was examined under light microscopic histochemistry and electron microscopy. To avoid loss of water-soluble glycogen during histological tissue preparation, fixation with ethanol and embedding in hydrophilic glycol methacrylate resin was used for light microscopy, and high-pressure freezing/freeze substitution for electron microscopy. Glycogen was detected on periodic acid-Schiff stain, periodic acid-thiosemicarbazide-silver proteinate reaction, and the digestion test with alpha-amylase. On embryonic day 14, glycogen began to accumulate in the proximal portions of the developing epithelial cords. On embryonic day 17, marked glycogen particles were seen at the basal portion of the ductal epithelial cells and an abrupt increase of glycogen accumulation occurred in the secretory cells in the terminal bulbs. Ultrastructural observation indicated large clumps of glycogen particles localized in the basal portion of the terminal bulb cells. The initiation of glycogen accumulation preceded the formation of lumens in the ducts and terminal bulbs. Furthermore, proliferation analysis by bromodeoxyuridine labeling showed that this glycogen accumulation followed the cessation of the epithelial cell proliferation. Postnatally, glycogen accumulation in the terminal bulbs became gradually inconspicuous and completely disappeared by postnatal day 3, but that in the ducts was retained until around postnatal day 12. Temporary glycogen accumulation after the cell proliferation and before/during the lumen formation and secretory granule formation suggests significant involvement of the carbohydrate metabolism in the organogenesis of the submandibular gland. PMID:17867343

  10. Human odontogenic epithelial cells derived from epithelial rests of Malassez possess stem cell properties.

    PubMed

    Tsunematsu, Takaaki; Fujiwara, Natsumi; Yoshida, Maki; Takayama, Yukihiro; Kujiraoka, Satoko; Qi, Guangying; Kitagawa, Masae; Kondo, Tomoyuki; Yamada, Akiko; Arakaki, Rieko; Miyauchi, Mutsumi; Ogawa, Ikuko; Abiko, Yoshihiro; Nikawa, Hiroki; Murakami, Shinya; Takata, Takashi; Ishimaru, Naozumi; Kudo, Yasusei

    2016-10-01

    Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of the Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. ERM cells are unique epithelial cells that remain in periodontal tissues throughout adult life. They have a functional role in the repair/regeneration of cement or enamel. Here, we isolated odontogenic epithelial cells from ERM in the periodontal ligament, and the cells were spontaneously immortalized. Immortalized odontogenic epithelial (iOdE) cells had the ability to form spheroids and expressed stem cell-related genes. Interestingly, iOdE cells underwent osteogenic differentiation, as demonstrated by the mineralization activity in vitro in mineralization-inducing media and formation of calcification foci in iOdE cells transplanted into immunocompromised mice. These findings suggest that a cell population with features similar to stem cells exists in ERM and that this cell population has a differentiation capacity for producing calcifications in a particular microenvironment. In summary, iOdE cells will provide a convenient cell source for tissue engineering and experimental models to investigate tooth growth, differentiation, and tumorigenesis. PMID:27479086

  11. Wound healing of intestinal epithelial cells

    PubMed Central

    Iizuka, Masahiro; Konno, Shiho

    2011-01-01

    The intestinal epithelial cells (IECs) form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs), regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing. PMID:21633524

  12. Invasive ductal carcinomas of the breast showing partial reversed cell polarity are associated with lymphatic tumor spread and may represent part of a spectrum of invasive micropapillary carcinoma.

    PubMed

    Acs, Geza; Esposito, Nicole N; Rakosy, Zsuzsa; Laronga, Christine; Zhang, Paul J

    2010-11-01

    Invasive micropapillary carcinomas (IMPC) of the breast are aggressive tumors frequently associated with lymphatic invasion and nodal metastasis even when micropapillary (MP) differentiation is very focal within the tumors. We have noticed that some breast carcinomas showing lymphatic spread but lacking histologic features of IMPC have occasional tumor cell clusters reminiscent of those of IMPC without the characteristic prominent retraction artifact. To study the clinicopathologic significance of such features, we prospectively selected 1323 invasive ductal carcinomas and determined the presence and extent of MP differentiation and retraction artifact in the tumors. One representative tumor block per case was used for immunostaining for epithelial membrane antigen (EMA). Partial reverse cell polarity (PRCP) was defined as prominent linear EMA reactivity on at least part of the periphery of tumor cell clusters usually associated with decreased cytoplasmic staining. The clinicopathologic features of carcinomas with PRCP were compared with IMPC and invasive ductal (no special type) carcinomas without this feature. Of the 1323 cases, 96 (7.3%) and 92 (7.0%) showed MP features and the presence of PRCP, respectively. We found that the presence of both PRCP and MP features were strongly associated with decreased cytoplasmic EMA immunoreactivity and the presence of lymphatic invasion and nodal metastasis, even if such features were present only very focally. Our results suggest that breast carcinomas with PRCP may have the same implication as MP differentiation and these tumors may represent part of a spectrum of IMPC. Complete or partial reversal of cell polarity may play a significant role in lymphatic tumor spread.

  13. Comparative evaluation of cancer stem cell markers in normal pancreas and pancreatic ductal adenocarcinoma.

    PubMed

    Vizio, Barbara; Mauri, Francesco A; Prati, Adriana; Trivedi, Pritesh; Giacobino, Alice; Novarino, Anna; Satolli, Maria Antonietta; Ciuffreda, Libero; Camandona, Michele; Gasparri, Guido; Bellone, Graziella

    2012-01-01

    Chemoresistance and self-renewal of cancer stem cells (CSC), found in many tumors including pancreatic ductal adenocarcinoma (PDAC), are believed to underlie tumor mass regrowth. The distribution of cells carrying the putative stem-cell markers CD133, Nestin, Notch1-4, Jagged1 and 2, ABCG2 and aldehyde dehydrogenase (ALDH1) was assessed immunohistochemically using PDAC and normal pancreas tissue microarrays. The immunoreactivity was semi-quantitatively graded against the normal pancreas and was correlated with the differentiation grade and disease stage. No statistical significant differences were found between normal pancreas and PDAC in the expression of Nestin, Notch1, 3 and 4, ABCG2 or ALDH1. Notch2 and Jagged1 and 2 expression were increased in PDAC. CD133-positive cells were above-normal in PDAC, but the difference was not statistically significant. Nestin, Notch1-4, Jagged1, ABCG2 and ALDH1 immunostaining scores were not correlated with tumor grade or disease stage. CD133 and Notch2 expression was significantly inversely correlated with tumor grade, but not disease stage. Notch3 immunostaining positively correlated with tumor stage, but not with differentiation grade. Jagged2 protein expression correlated inversely with disease stage, but not with tumor grade. From the clinical standpoint, improved delineation of the tumor CSC signature, putatively responsible for tumor initiation and recurrence after initial response to chemotherapy, may offer novel therapeutic targets for this highly lethal cancer.

  14. Lifespan Extension and Sustained Expression of Stem Cell Phenotype of Human Breast Epithelial Stem Cells in a Medium with Antioxidants

    PubMed Central

    Wang, Kai-Hung; Kao, An-Pei; Chang, Chia-Cheng; Lin, Ta-Chin

    2016-01-01

    We have previously reported the isolation and culture of a human breast epithelial cell type with stem cell characteristics (Type I HBEC) from reduction mammoplasty using the MSU-1 medium. Subsequently, we have developed several different normal human adult stem cell types from different tissues using the K-NAC medium. In this study, we determined whether this low calcium K-NAC medium with antioxidants (N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate) is a better medium to grow human breast epithelial cells. The results clearly show that the K-NAC medium is a superior medium for prolonged growth (cumulative population doubling levels ranged from 30 to 40) of normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures. Thus, this new method of growing Type I HBECs will be very useful in future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy. PMID:27807451

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

    EPA Science Inventory

    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...

  16. Replication of cultured lung epithelial cells

    SciTech Connect

    Guzowski, D.; Bienkowski, R.

    1986-03-05

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to (/sup 3/H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems.

  17. Control of the epithelial stem cell epigenome: the shaping of epithelial stem cell identity.

    PubMed

    Iglesias-Bartolome, Ramiro; Callejas-Valera, Juan Luis; Gutkind, J Silvio

    2013-04-01

    The squamous epithelium covering the skin and oral mucosa relies on epithelial stem cells for tissue renewal. Dynamic changes in DNA methylation, histone methylation and acetylation, and higher order chromatin structure are required to preserve their self-renewal capacity while orchestrating the timely execution of cell differentiation programs. This complex network of epigenetic modifications shapes the epithelial stem cell identity and fate. Pathological alterations can be perceived by aberrant chromatin sensors, such as the INK4/ARF locus, which initiate tumor suppressive cell senescence programs, and can often result in epithelial stem cell exhaustion. Unveiling the mechanisms controlling the epigenome in epithelial stem cells may help protect against the loss of their tissue regenerative capacity, thereby preventing premature aging without increasing cancer risk. PMID:23434069

  18. Pleiotrophin (PTN) expression and function and in the mouse mammary gland and mammary epithelial cells.

    PubMed

    Rosenfield, Sonia M; Bowden, Emma T; Cohen-Missner, Shani; Gibby, Krissa A; Ory, Virginie; Henke, Ralf T; Riegel, Anna T; Wellstein, Anton

    2012-01-01

    Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.

  19. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

    PubMed Central

    Ricci, Claudio; Mota, Carlos; Moscato, Stefania; D’Alessandro, Delfo; Ugel, Stefano; Sartoris, Silvia; Bronte, Vincenzo; Boggi, Ugo; Campani, Daniela; Funel, Niccola; Moroni, Lorenzo; Danti, Serena

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs. PMID:25482337

  20. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models.

    PubMed

    Ricci, Claudio; Mota, Carlos; Moscato, Stefania; D'Alessandro, Delfo; Ugel, Stefano; Sartoris, Silvia; Bronte, Vincenzo; Boggi, Ugo; Campani, Daniela; Funel, Niccola; Moroni, Lorenzo; Danti, Serena

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

  1. Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma.

    PubMed

    Jablonska, Karolina; Pula, Bartosz; Zemla, Agata; Owczarek, Tomasz; Wojnar, Andrzej; Rys, Janusz; Ambicka, Aleksandra; Podhorska-Okolow, Marzena; Ugorski, Maciej; Dziegiel, Piotr

    2013-04-01

    In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonin's oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA-MB-231 compared with ER+ cell lines MCF-7 and SK-BR-3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients' longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event-free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.

  2. Protons sensitize epithelial cells to mesenchymal transition.

    PubMed

    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.

  3. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

    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. PMID:22844446

  4. SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells

    PubMed Central

    2010-01-01

    Background The vertebrate pancreas contains islet, acinar and ductal cells. These cells derive from a transient pool of multipotent pancreatic progenitors during embryonic development. Insight into the genetic determinants regulating pancreatic organogenesis will help the development of cell-based therapies for the treatment of diabetes mellitus. Suppressor enhancer lin12/Notch 1 like (Sel1l) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by Sel1l remain elusive. Results We have characterized the pancreatic phenotype of mice carrying a gene trap mutation in Sel1l. We show that Sel1l expression in the developing pancreas coincides with differentiation of the endocrine and exocrine lineages. Mice homozygous for the gene trap mutation die prenatally and display an impaired pancreatic epithelial morphology and cell differentiation. The pancreatic epithelial cells of Sel1l mutant embryos are confined to the progenitor cell state throughout the secondary transition. Pharmacological inhibition of Notch signaling partially rescues the pancreatic phenotype of Sel1l mutant embryos. Conclusions Together, these data suggest that Sel1l is essential for the growth and differentiation of endoderm-derived pancreatic epithelial cells during mouse embryonic development. PMID:20170518

  5. Respiratory epithelial cells orchestrate pulmonary innate immunity

    PubMed Central

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and ‘instruct’ the professional immune system to protect the lungs from infection and injury. PMID:25521682

  6. Classifying the Progression of Ductal Carcinoma from Single-Cell Sampled Data via Integer Linear Programming: A Case Study.

    PubMed

    Catanzaro, Daniele; Shackney, Stanley E; Schaffer, Alejandro A; Schwartz, Russell

    2016-01-01

    Ductal Carcinoma In Situ (DCIS) is a precursor lesion of Invasive Ductal Carcinoma (IDC) of the breast. Investigating its temporal progression could provide fundamental new insights for the development of better diagnostic tools to predict which cases of DCIS will progress to IDC. We investigate the problem of reconstructing a plausible progression from single-cell sampled data of an individual with synchronous DCIS and IDC. Specifically, by using a number of assumptions derived from the observation of cellular atypia occurring in IDC, we design a possible predictive model using integer linear programming (ILP). Computational experiments carried out on a preexisting data set of 13 patients with simultaneous DCIS and IDC show that the corresponding predicted progression models are classifiable into categories having specific evolutionary characteristics. The approach provides new insights into mechanisms of clonal progression in breast cancers and helps illustrate the power of the ILP approach for similar problems in reconstructing tumor evolution scenarios under complex sets of constraints.

  7. Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition

    PubMed Central

    Wang, Jehng-Kang; Lee, Ming-Shyue; Tseng, I-Chu; Chou, Feng-Pai; Chen, Ya-Wen; Fulton, Amy; Lee, Herng-Sheng; Chen, Cheng-Jueng; Johnson, Michael D.; Lin, Chen-Yong

    2009-01-01

    Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integrity of, the epithelium. Matriptase is synthesized as a zymogen and undergoes autoactivation to become an active protease that is immediately inhibited by, and forms complexes with, hepatocyte growth factor activator inhibitor (HAI-1). To investigate where matriptase is activated and how it is secreted in vivo, we determined the expression and activation status of matriptase in seminal fluid and urine and the distribution and subcellular localization of the protease in the prostate and kidney. The in vivo studies revealed that while the latent matriptase is localized at the basolateral surface of the ductal epithelial cells of both organs, only matriptase-HAI-1 complexes and not latent matriptase are detected in the body fluids, suggesting that activation, inhibition, and transcytosis of matriptase would have to occur for the secretion of matriptase. These complicated processes involved in the in vivo secretion were also observed in polarized Caco-2 intestinal epithelial cells. The cells target latent matriptase to the basolateral plasma membrane where activation, inhibition, and secretion of matriptase appear to take place. However, a proportion of matriptase-HAI-1 complexes, but not the latent matriptase, appears to undergo transcytosis to the apical plasma membrane for secretion. When epithelial cells lose their polarity, they secrete both latent and activated matriptase. Although most epithelial cells retain very low levels of matriptase-HAI-1 complex by rapidly secreting the complex, gastric chief cells may activate matriptase and store matriptase-HAI-1 complexes in the pepsinogen-secretory granules, suggesting an intracellular activation and regulated secretion in these cells. Taken together, while zymogen activation and closely coupled HAI-1-mediated inhibition are common features for matriptase regulation, the cellular location of matriptase activation and

  8. Functional expression of TLR5 in murine salivary gland epithelial cells.

    PubMed

    Iwasa, Satoko; Ota, Hirotaka; Nishio, Kensuke; Ohtsu, Mariko; Kusunoki, Masafumi; Gojoubori, Takahiro; Shirakawa, Tetsuo; Asano, Masatake

    2016-01-01

    Toll-like receptors (TLR) recognize microbe-associated molecular patterns and induce the innate immune response. Among them, TLR5 recognizes the Gram-negative bacterial component flagellin. The aim of this study was to examine the expression of TLR5 in mouse salivary gland (SG). The SG was excised from 8- to 10-week-old female C57BL/6 mice. Salivary gland epithelial cells (SGECs) were purified and subjected to reverse transcription polymerase chain reaction (RT-PCR). Western blotting was performed to detect TLR5 expression at the protein level in several organs. The localization of TLR5 in SG was examined using immunohistochemical staining. The responsiveness of SGECs to flagellin was further examined by evaluating the induction of CXCL1 by real-time PCR and immunoprecipitation followed by Western blotting. TLR5 expression in SG was confirmed at the gene and protein levels. Immunohistochemical staining detected TLR5 in both acinic and ductal cells of the sublingual gland, but not in serous acinic cells of the submandibular gland. Although TLR5 was detected throughout the cytoplasm in ductal cells, positive staining was observed on the basal side of the mucous acinic cells. The purified SGECs responded to flagellin and induced the production of CXCL1. These findings suggest that TLR5 is functionally expressed in the SG and responds to its cognate ligand flagellin. (J Oral Sci 58, 317-323, 2016). PMID:27665969

  9. Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    PubMed Central

    Muir, Amanda B.; Dods, Kara; Noah, Yuli; Toltzis, Sarit; Chandramouleeswaran, Prasanna Modayur; Lee, Anna; Benitez, Alain; Bedenbaugh, Adam; Falk, Gary W.; Wells, Rebecca G.; Nakagawa, Hiroshi; Wang, Mei-Lun

    2015-01-01

    Background and Aims Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. Methods and Results Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. Conclusions Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis. PMID:25183431

  10. Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

    SciTech Connect

    Booth, Brian W.; Boulanger, Corinne A.; Anderson, Lisa H.; Jimenez-Rojo, Lucia; Brisken, Cathrin; Smith, Gilbert H.

    2010-02-01

    Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D {beta}-geo (CD{beta}geo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CD{beta}geo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG{sup -/-} mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.

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

    SciTech Connect

    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.

  12. Growth inhibition of Candida by human oral epithelial cells.

    PubMed

    Steele, C; Leigh, J; Swoboda, R; Fidel, P L

    2000-11-01

    Oropharyngeal candidiasis (OPC) caused by Candida albicans is a significant problem in human immunodeficiency virus (HIV)-infected persons. Recognizing the paucity of information on innate and/or adaptive mucosal host defenses against C. albicans, we recently reported that human and nonhuman primate and mouse vaginal epithelial cells inhibit the growth of C. albicans in vitro. In the present study, oral epithelial cells collected from saliva of healthy volunteers and a purified oral epithelial cell line were found to inhibit blastoconidia and/or hyphal growth of several Candida species. Cell contact was a strict requirement for the epithelial cell anti-Candida activity; neither saliva nor culture supernatants alone inhibited Candida growth, and addition of saliva to the coculture did not modulate the epithelial cell activity. Finally, epithelial cell anti-Candida activity was significantly lower in HIV-infected persons with OPC. Together, these results suggest that oral epithelial cells may play a role in innate resistance against OPC.

  13. A comparison study of pancreatic acinar cell carcinoma with ductal adenocarcinoma using computed tomography in Chinese patients

    PubMed Central

    Wang, Qingbing; Wang, Xiaolin; Guo, Rongfang; Li, Guoping

    2016-01-01

    Pancreatic acinar cell carcinoma (ACC) is a rare tumor that is difficult to diagnose preoperatively. The aim of this study was to evaluate and describe the computed tomography (CT) features of ACC and compare the results with pancreatic ductal adenocarcinoma (DAC) for improving preoperative diagnosis. The control group consisted of 34 patients with DAC collected from the pathology electronic database. The CT imaging from nine patients with pathologically confirmed ACC was retrospectively reviewed. Two radiologists independently assessed the tumor location, size, texture, and enhancement patterns. We found that 64.3% (9/14) of ACC tumors were homogeneous and 35.7% (5/14) had necrosis. The percentage of common bile duct and pancreatic ductal dilation was 14.3% (2/14) and 7.1% (1/14), respectively. The mean size of ACC was 50.1±24.2 mm. The mean attenuation of ACC was 35.4±3.9 Hounsfield unit (HU) before enhancement, 73.1±42.9 HU in arterial phase, and 71.8±15.6 HU in port venous phase. It is difficult to distinguish ACC from DAC preoperatively only based on CT findings. However, compared with DAC, we found that ACC tumors are likely to be larger and contain more heterogeneous intratumoral necrotic hypovascular regions, and less pancreatic ductal and common biliary dilation. PMID:27660464

  14. A comparison study of pancreatic acinar cell carcinoma with ductal adenocarcinoma using computed tomography in Chinese patients

    PubMed Central

    Wang, Qingbing; Wang, Xiaolin; Guo, Rongfang; Li, Guoping

    2016-01-01

    Pancreatic acinar cell carcinoma (ACC) is a rare tumor that is difficult to diagnose preoperatively. The aim of this study was to evaluate and describe the computed tomography (CT) features of ACC and compare the results with pancreatic ductal adenocarcinoma (DAC) for improving preoperative diagnosis. The control group consisted of 34 patients with DAC collected from the pathology electronic database. The CT imaging from nine patients with pathologically confirmed ACC was retrospectively reviewed. Two radiologists independently assessed the tumor location, size, texture, and enhancement patterns. We found that 64.3% (9/14) of ACC tumors were homogeneous and 35.7% (5/14) had necrosis. The percentage of common bile duct and pancreatic ductal dilation was 14.3% (2/14) and 7.1% (1/14), respectively. The mean size of ACC was 50.1±24.2 mm. The mean attenuation of ACC was 35.4±3.9 Hounsfield unit (HU) before enhancement, 73.1±42.9 HU in arterial phase, and 71.8±15.6 HU in port venous phase. It is difficult to distinguish ACC from DAC preoperatively only based on CT findings. However, compared with DAC, we found that ACC tumors are likely to be larger and contain more heterogeneous intratumoral necrotic hypovascular regions, and less pancreatic ductal and common biliary dilation.

  15. A comparison study of pancreatic acinar cell carcinoma with ductal adenocarcinoma using computed tomography in Chinese patients.

    PubMed

    Wang, Qingbing; Wang, Xiaolin; Guo, Rongfang; Li, Guoping

    2016-01-01

    Pancreatic acinar cell carcinoma (ACC) is a rare tumor that is difficult to diagnose preoperatively. The aim of this study was to evaluate and describe the computed tomography (CT) features of ACC and compare the results with pancreatic ductal adenocarcinoma (DAC) for improving preoperative diagnosis. The control group consisted of 34 patients with DAC collected from the pathology electronic database. The CT imaging from nine patients with pathologically confirmed ACC was retrospectively reviewed. Two radiologists independently assessed the tumor location, size, texture, and enhancement patterns. We found that 64.3% (9/14) of ACC tumors were homogeneous and 35.7% (5/14) had necrosis. The percentage of common bile duct and pancreatic ductal dilation was 14.3% (2/14) and 7.1% (1/14), respectively. The mean size of ACC was 50.1±24.2 mm. The mean attenuation of ACC was 35.4±3.9 Hounsfield unit (HU) before enhancement, 73.1±42.9 HU in arterial phase, and 71.8±15.6 HU in port venous phase. It is difficult to distinguish ACC from DAC preoperatively only based on CT findings. However, compared with DAC, we found that ACC tumors are likely to be larger and contain more heterogeneous intratumoral necrotic hypovascular regions, and less pancreatic ductal and common biliary dilation. PMID:27660464

  16. NOGGIN IS REQUIRED FOR NORMAL LOBE PATTERNING AND DUCTAL BUDDING IN THE MOUSE PROSTATE

    PubMed Central

    Cook, Crist; Vezina, Chad M.; Hicks, Sarah M.; Shaw, Aubie; Yu, Min; Peterson, Richard E.; Bushman, Wade

    2008-01-01

    Mesenchymal expression of the BMP antagonist NOGGIN during prostate development plays a critical role in pre-natal ventral prostate development and opposes BMP4-mediated inhibition of cell proliferation during postnatal ductal development. Morphologic examination of newborn Noggin-/- male fetuses revealed genitourinary anomalies including cryptorchidism, incomplete separation of the hindgut from the urogenital sinus (UGS), absence of the ventral mesenchymal pad and a complete loss of ventral prostate (VP) budding. Examination of lobe-specific marker expression in the E14 Noggin-/- UGS rescued by transplantation under the renal capsule of a male nude mouse confirmed a complete loss of VP determination. More modest effects were observed in the other lobes, including decreased number of ductal buds in the dorsal and lateral prostates of newborn Noggin-/- males. BMP4 and BMP7 have been shown to inhibit ductal budding and outgrowth by negatively regulating epithelial cell proliferation. We show here that NOGGIN can neutralize budding inhibition by BMP4 and rescues branching morphogenesis of BMP4-exposed UGS in organ culture and show that the effects of BMP4 and NOGGIN activities converge on P63+ epithelial cells located at nascent duct tips. Together, these studies show that the BMP-NOGGIN axis regulates patterning of the ventral prostate, regulates ductal budding, and controls proliferation of P63+ epithelial cells in the nascent ducts of developing mouse prostate. PMID:18028901

  17. Chronic inflammatory cells with epithelial cell characteristics in teleost fishes.

    PubMed

    Noga, E J; Dykstra, M J; Wright, J F

    1989-09-01

    Certain cells that participate in the chronic inflammatory response of teleost fishes have many features typical of epithelioid cells of mammals. Such features include high metabolic activity, frequent phagolysosomes, and cytoplasmic interdigitations between adjacent cells; however, the epithelioid granulomas formed in response to certain diseases in teleost fishes also have several features associated with epithelial cells. Cases of ulcerative mycosis or acid-fast bacterial infection in Atlantic menhaden (Brevoortia tyrannus), fungal infection in silver perch (Bairdiella chrysoura), and mycobacteriosis in Mozambique tilapia (Oreochromis mossambicus) had epithelioid cells that were joined together by well-formed desmosomes with tonofilaments. "Mature granulomas" of the ulcerative mycosis-infected menhaden stained positively for cytokeratin, a cytoskeletal protein that is considered to be highly specific for epithelial cells. The consistent presence of these heretofore unrecognized epithelial features suggest that they may be characteristic of certain types of cells participating in piscine chronic inflammation. PMID:2686148

  18. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  19. Desialylation of Spermatozoa and Epithelial Cell Glycocalyx Is a Consequence of Bacterial Infection of the Epididymis.

    PubMed

    Khosravi, Farhad; Michel, Vera; Galuska, Christina E; Bhushan, Sudhanshu; Christian, Philipp; Schuppe, Hans-Christian; Pilatz, Adrian; Galuska, Sebastian P; Meinhardt, Andreas

    2016-08-19

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis. PMID:27339898

  20. Collision of Ductal Carcinoma In Situ of Anogenital Mammary-like Glands and Vulvar Sarcomatoid Squamous Cell Carcinoma.

    PubMed

    Tran, Tien A N; Deavers, Michael T; Carlson, J Andrew; Malpica, Anais

    2015-09-01

    A spectrum of invasive adenocarcinomas presumably arising from the anogenital mammary-like glands of the vulva has been reported. Even rarer are the cases of pure ductal carcinoma in situ that originated from these unique glandular structures. Herein, we report an 81-yr-old woman presented with an invasive well-differentiated squamous cell carcinoma of the vulva. Unexpectedly, the underlying dermis demonstrated a cystically dilated structure that displayed a layer of malignant squamous cells in the periphery, and a second centrally located population of neoplastic cells exhibiting glandular differentiation. In addition, a spindle and pleomorphic malignant cell population consistent with a sarcomatoid carcinoma was identified around the cystic structure. Scattered benign anogenital mammary-like glands were present in the adjacent dermis. The histologic and immunohistochemical findings were consistent with those of vulvar squamous cell carcinoma that has undergone sarcomatoid transformation after spreading in a pagetoid fashion into an underlying focus of ductal carcinoma in situ of anogenital mammary-like gland origin.

  1. Detection of Bone Marrow Derived Lung Epithelial Cells

    PubMed Central

    Kassmer, Susannah H.; Krause, Diane S.

    2010-01-01

    Studies on the ability of bone marrow derived cells to adopt the morphology and protein expression of epithelial cells in vivo have expanded rapidly over the last decade, and hundreds of publications report that bone marrow derived cells can become epithelial cells of multiple organs including lung, liver, GI tract, skin, pancreas and others. In this review, we critically evaluate the literature related to engraftment of bone marrow derived cells as epithelial cells in the lung. Over 40 manuscripts focused on whether bone marrow cells can differentiate into lung epithelial cells have been published, nearly all of which claim to identify marrow derived epithelial cells. A few investigations have concluded that no such cells are present and that the phenomenon of marrow derived epithelial cells is based on detection artifacts. Here we discuss the problems that exist in published papers identifying marrow derived epithelial cells, and propose standards for detection methods that provide the most definitive data. Identification of BM derived epithelial cells requires reliable and sensitive techniques for their detection, which must include cell identification based on the presence of an epithelial marker and the absence of blood cell markers as well as a marker for donor BM origin. In order for these studies to be rigorous, they must also use approaches to rule out cell overlap by microscopy or single cell isolation. Once these stringent criteria for identification of marrow derived epithelial cells are used universally, then the field can move forward to address the critical questions regarding which bone marrow derived cells are responsible for engraftment as epithelial cells, the mechanisms by which this occurs, whether these cells play a role in normal tissue repair, and whether specific cell subsets can be used for therapeutic benefit. PMID:20447442

  2. Human mesenchymal stem cells cultured with salivary gland biopsies adopt an epithelial phenotype.

    PubMed

    Maria, Ola M; Tran, Simon D

    2011-06-01

    Sjogren's syndrome and radiotherapy for head and neck cancer result in severe xerostomia and irreversible salivary gland damage for which no effective treatment is currently available. Cell culture methods of primary human salivary gland epithelial cells (huSGs) are slow and cannot provide a sufficient number of cells. In addition, the majority of cultured huSGs are of a ductal phenotype and thus not fluid/saliva secretory cells. Some reports indicated that mesenchymal stem cells (MSCs) possessed the potential to differentiate into epithelial cells. To test this hypothesis with huSGs, a coculture system containing 2 chambers separated by a polyester membrane was used to study the capacity of human MSCs to adopt an epithelial phenotype when cocultured with human salivary gland biopsies. Results were that 20%-40% of cocultured MSCs expressed tight junction proteins [claudin-1 (CLDN-1), -2, -3, and -4; occludin; junctional adhesion molecule-A; and zonula occludens-1] as well as other epithelial markers [aquaporin-5, α-amylase (α-AMY), and E-cadherin], and generated a higher transepithelial electrical resistance. Electron microscopy demonstrated that these MSCs had comparable cellular structures to huSGs, such as tight junction structures and numerous secretory granules. Quantitative real time (RT)-polymerase chain reaction revealed an upregulation of several salivary genes (aquaporin-5, AMY, and CLDN-2). Moreover, the amounts of α-AMY detected in cocultured MSCs were comparable to those detected in huSGs control cultures. These data suggest that cocultured MSCs can demonstrate a temporary change into a salivary gland acinar phenotype.

  3. Isolated Epithelial Cells of the Toad Bladder

    PubMed Central

    Gatzy, J. T.; Berndt, W. O.

    1968-01-01

    Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. QOO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O2 at a rate of 4 µl hr-1 dry wt-1. QOO2 was increased 50% by ADH (100 U/liter) or by cyclic 3',5'-AMP (10 mM/liter). Na+-free Ringer's depressed the QOO2 by 40%. The QOO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na+-free Ringer's but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na+ lack or ADH. The intracellular Na+ and K+ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H2O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na+ concentrations. Cells unresponsive to ADH and Na+ lack had high sucrose spaces and low transcellular membrane gradients of Na+, K+, and Cl-. The results suggest that trypsin and EDTA disaggregation damage the active Na+ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally. PMID:4300150

  4. Scrib is Required for Epithelial Cell Identity and Prevents Epithelial To Mesenchymal Transition in the Mouse

    PubMed Central

    Yamben, Idella F.; Rachel, Rivka A.; Shatadal, Shalini; Copeland, Neal G.; Jenkins, Nancy A.; Warming, Soren; Griep, Anne E.

    2013-01-01

    The integrity and function of epithelial tissues depends on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling. PMID:24095903

  5. Interaction with epithelial cells modifies airway macrophage response to ozone.

    PubMed

    Bauer, Rebecca N; Müller, Loretta; Brighton, Luisa E; Duncan, Kelly E; Jaspers, Ilona

    2015-03-01

    The initial innate immune response to ozone (O3) 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 signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

  6. Minor Salivary Gland Changes in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma - A Histopathological Study

    PubMed Central

    Chitturi, Ravi Teja; Ragunathan, Yoithapprabhunath Thukanayakanpalayam; Lakshmi, Suman Jhansi; Nallusamy, Jaisanghar; Joseph, Isaac

    2016-01-01

    Introduction The most common etiology for Oral Squamous Cell Carcinoma (OSCC) is tobacco and tobacco related products which cause nuclear damage to the keratinocytes. The chemical carcinogens not only affect the lining of oral epithelium but also affect the lining epithelium of the excretory ducts of the salivary glands. Thus, there is a possibility of epithelial dysplasia of the salivary duct epithelium which may lead to potential malignant transformation. Aim The study was performed to see the changes in the minor salivary glands and excretory ducts in cases of oral epithelial dysplasia and OSCC. Materials and Methods A total of 278 archival cases of mild, moderate and severe epithelial dysplasia, carcinoma in situ, OSCC including verrucous carcinoma were histopathologically evaluated to observe changes in the excretory ducts and the minor salivary glands. Results In the study there were 56.5% males and 43.5% females. The age group that was most commonly affected in both the sexes was 50-60 yr old. Buccal mucosa was the most common site of involvement. Ductal changes observed in the excretory duct include simple hyperplasia, metaplastic changes such as mucous, oncocytic & squamous, and infiltration of inflammatory cells and malignant cells. Acinar changes observed were degeneration, squamous metaplasia, myoepithelial cell proliferation and inflammatory cell infiltration. Both the excretory ducts and ducts within the gland showed dysplasia. Conclusion According to observations in our study it is suggested that histopathological interpretation for oral mucosal lesions especially oral epithelial dysplasias and OSCC should also include changes related to salivary gland tissue to provide a better treatment plan and prevent recurrence of the malignant tumours. PMID:27630945

  7. Minor Salivary Gland Changes in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma - A Histopathological Study

    PubMed Central

    Chitturi, Ravi Teja; Ragunathan, Yoithapprabhunath Thukanayakanpalayam; Lakshmi, Suman Jhansi; Nallusamy, Jaisanghar; Joseph, Isaac

    2016-01-01

    Introduction The most common etiology for Oral Squamous Cell Carcinoma (OSCC) is tobacco and tobacco related products which cause nuclear damage to the keratinocytes. The chemical carcinogens not only affect the lining of oral epithelium but also affect the lining epithelium of the excretory ducts of the salivary glands. Thus, there is a possibility of epithelial dysplasia of the salivary duct epithelium which may lead to potential malignant transformation. Aim The study was performed to see the changes in the minor salivary glands and excretory ducts in cases of oral epithelial dysplasia and OSCC. Materials and Methods A total of 278 archival cases of mild, moderate and severe epithelial dysplasia, carcinoma in situ, OSCC including verrucous carcinoma were histopathologically evaluated to observe changes in the excretory ducts and the minor salivary glands. Results In the study there were 56.5% males and 43.5% females. The age group that was most commonly affected in both the sexes was 50-60 yr old. Buccal mucosa was the most common site of involvement. Ductal changes observed in the excretory duct include simple hyperplasia, metaplastic changes such as mucous, oncocytic & squamous, and infiltration of inflammatory cells and malignant cells. Acinar changes observed were degeneration, squamous metaplasia, myoepithelial cell proliferation and inflammatory cell infiltration. Both the excretory ducts and ducts within the gland showed dysplasia. Conclusion According to observations in our study it is suggested that histopathological interpretation for oral mucosal lesions especially oral epithelial dysplasias and OSCC should also include changes related to salivary gland tissue to provide a better treatment plan and prevent recurrence of the malignant tumours.

  8. Ouabain modulates ciliogenesis in epithelial cells

    PubMed Central

    Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Contreras, Ruben G.; Galvan, Ivan; Muñoz-Estrada, Jesus; Cereijido, Marcelino

    2011-01-01

    The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life. PMID:22143774

  9. [Research progress of corneal epithelial basal cells and basement membrane].

    PubMed

    Qu, J H; Sun, X G

    2016-09-11

    The cylinder cells at the bottom of corneal epithelial cells are basal cells. Their cytoplasm contains keratin intermediate filament which is important in secretion of basement membrane. Corneal epithelial dysfunction due to diabetes or ocular surgery is intimately related with basal cell abnormality. Corneal epithelial basement membrane is a highly specific extracellular matrix which is made up of lamina lucida and lamina densa. It plays an extremely important role in renewal and restoration. Many ocular abnormalities and diseases have been described to relate to the corneal epithelial basement membrane, such as traumatic recurrent corneal erosion, corneal dystrophy and keratoconus. (Chin J Ophthalmol, 2016, 52: 703-707). PMID:27647251

  10. Slug inhibits pancreatic cancer initiation by blocking Kras-induced acinar-ductal metaplasia

    PubMed Central

    Ebine, Kazumi; Chow, Christina R.; DeCant, Brian T.; Hattaway, Holly Z.; Grippo, Paul J.; Kumar, Krishan; Munshi, Hidayatullah G.

    2016-01-01

    Cells in the pancreas that have undergone acinar-ductal metaplasia (ADM) can transform into premalignant cells that can eventually become cancerous. Although the epithelial-mesenchymal transition regulator Snail (Snai1) can cooperate with Kras in acinar cells to enhance ADM development, the contribution of Snail-related protein Slug (Snai2) to ADM development is not known. Thus, transgenic mice expressing Slug and Kras in acinar cells were generated. Surprisingly, Slug attenuated Kras-induced ADM development, ERK1/2 phosphorylation and proliferation. Co-expression of Slug with Kras also attenuated chronic pancreatitis-induced changes in ADM development and fibrosis. In addition, Slug attenuated TGF-α-induced acinar cell metaplasia to ductal structures and TGF-α-induced expression of ductal markers in ex vivo acinar explant cultures. Significantly, blocking the Rho-associated protein kinase ROCK1/2 in the ex vivo cultures induced expression of ductal markers and reversed the effects of Slug by inducing ductal structures. In addition, blocking ROCK1/2 activity in Slug-expressing Kras mice reversed the inhibitory effects of Slug on ADM, ERK1/2 phosphorylation, proliferation and fibrosis. Overall, these results increase our understanding of the role of Slug in ADM, an early event that can eventually lead to pancreatic cancer development. PMID:27364947

  11. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells

    PubMed Central

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C.; Liu, Jinsong

    2016-01-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  12. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells.

    PubMed

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C; Liu, Jinsong

    2016-03-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  13. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  14. Characterization of glomerular epithelial cell matrix receptors.

    PubMed Central

    Adler, S.

    1992-01-01

    Integrin matrix receptors on glomerular epithelial cells (GEC) may play an important role in adhesion of GEC to the glomerular basement membrane (GBM) and in the maintenance of normal glomerular permeability. Therefore, the author determined the types of matrix receptors present on cultured rat GEC and examined their interactions with several components of the extracellular matrix. Beta 1 integrin matrix receptors were detected on all three glomerular cell types in rat kidney in vivo and at areas of cell-cell contact on cultured GEC. Glomerular epithelial cell adhesion to types I and IV collagen was slightly greater than to laminin and fibronectin. Adhesion to fibronectin was significantly inhibited by a synthetic peptide containing the RGD adhesion sequence. Immunoprecipitation of lysates of surface-iodinated GEC showed the presence of alpha 3 beta 1 integrin. Chromatography of lysates on immobilized collagen showed alpha 3 beta 1 integrin and a 70- to 75-kd protein band as the collagen receptors on GEC. Chromatography on the 120-kd cell-binding fragment of fibronectin disclosed only alpha 3 beta 1 as a specific fibronectin receptor. Antibody to the beta 1 integrin chain inhibited adhesion to laminin and collagen. These studies demonstrate that in vitro, as in vivo, GEC appear to express only alpha 3 beta 1 integrin. Furthermore, this matrix receptor is capable of mediating GEC adhesion to collagen, fibronectin, and laminin, components of the GBM, and presumably plays a similar role in promoting GEC adhesion to GBM in vivo. Images Figure 1 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1325740

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

    SciTech Connect

    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.

  16. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

    SciTech Connect

    Lochter, A.; Galosy, S.; Muschler, J.; Freedman, N.; Werb, Z.; Bissell, M.J.

    1997-08-11

    Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

  17. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    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.

  18. Anthrax Toxin Entry into Polarized Epithelial Cells

    PubMed Central

    Beauregard, Kathryn E.; Wimer-Mackin, Susan; Collier, R. John; Lencer, Wayne I.

    1999-01-01

    We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl− secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl− secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease. PMID:10338515

  19. Silk Film Topography Directs Collective Epithelial Cell Migration

    PubMed Central

    Rosenblatt, Mark I.

    2012-01-01

    The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

  20. Ascl3 expression marks a progenitor population of both acinar and ductal cells in mouse salivary glands.

    PubMed

    Bullard, Tara; Koek, Laurie; Roztocil, Elisa; Kingsley, Paul D; Mirels, Lily; Ovitt, Catherine E

    2008-08-01

    Ascl3, also know as Sgn1, is a member of the mammalian achaete scute (Mash) gene family of transcription factors, which have been implicated in cell fate specification and differentiation. In the mouse salivary gland, expression of Ascl3 is restricted to a subset of duct cells. Salivary gland function depends on the secretory acinar cells, which are responsible for saliva formation, and duct cells, which modify the saliva and conduct it to the oral cavity. The salivary gland ducts are also the putative site of progenitor cells in the adult gland. Using a Cre recombinase-mediated reporter system, we followed the fate of Ascl3-expressing cells after the introduction of an EGFP-Cre expression cassette into the Ascl3 locus by homologous recombination. Lineage tracing shows that these cells are progenitors of both acinar and ductal cell types in all three major salivary glands. In the differentiated progeny, expression of Ascl3 is down-regulated. These data directly demonstrate a progenitor-progeny relationship between duct cells and the acinar cell compartment, and identify a population of multipotent progenitor cells, marked by expression of Ascl3, which is capable of generating both gland cell types. We conclude that Ascl3-expressing cells contribute to the maintenance of the adult salivary glands.

  1. Detection of ductal dysplasia in mammary outgrowths derived from carcinogen-treated virgin female BALB/c mice

    SciTech Connect

    Ethier, S.P.; Ullrich, R.L.

    1982-05-01

    These studies were undertaken to determine if altered growth potential of mammary epithelial cells could be detected in outgrowths derived from monodispersed mammary cells of virgin female BALB/c mice previously exposed to ionizing radiation or 7, 12-dimethylbenz(a)anthracene (DMBA). Twenty-four hr prior to cell dissociation, donor animals were exposed to either 100 rads of gamma-ray irradiation, 0.25 mg of DMBA, or 0.075 mg of DMBA. Control donors were untreated. Mammary outgrowths were then derived from these donor cells by injecting either 10(5) or 10(4) cells into the gland-free mammary fat pads of three-week-old virgin female BALB/c mice. Mammary outgrowths were classified either as having a normal ductal architecture or as having ductal dysplasia. Ductal dysplasias were further classified on the basis of an index of severity, which was an arbitrary index based on the number of abnormal ductal structures within each lesion. The data indicated that treatment of donor animals with either gamma-radiation or DMBA increased the frequency of ductal lesions over control levels; however, both the frequency and severity of the lesions depended on the number of cells which were injected into the fat pad. The data indicated that ductal dysplasias were more common and more severe in outgrowths derived 10(4) rather than 10(5) cells. The ductal lesions observed in this study resembled both morphologically and histologically ductal abnormalities which have been associated with the pathogenesis of mammary carcinoma in both rats and mice.

  2. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

    PubMed Central

    Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

    2012-01-01

    Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

  3. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  4. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

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

    PubMed Central

    Bauer, Rebecca N.; Müller, Loretta; Brighton, Luisa E.; Duncan, Kelly E.

    2015-01-01

    The initial innate immune response to ozone (O3) 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 signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell–Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell–derived signals are important determinants of Mac immunophenotype and response to O3. PMID:25054807

  6. Eikenella corrodens adherence to human buccal epithelial cells.

    PubMed Central

    Yamazaki, Y; Ebisu, S; Okada, H

    1981-01-01

    The mechanism of Eikenella corrodens adherence to human buccal epithelial cells in vitro was studied. Initial experiments to determine the optimal conditions for adherence of E. corrodens to buccal epithelial cells showed that adherence was dependent on time, temperature, bacterial concentration, and pH. Different strains of E. corrodens varied in their ability to adhere, and strain 1073 showed the greatest ability in adherence. Strain 1073 was selected for studies of adherence mechanisms. Trypsin treatment or heating (100 degrees C, 10 min) of the bacterial cells abolished their capacity to adhere to buccal epithelial cells. Treatment of buccal epithelial cells with trypsin also abolished adherence of E. corrodens 1073, whereas neuraminidase treatment of buccal epithelial cells enhanced the adherence. The adherence was inhibited by ethylenediaminetetraacetic acid and restored by adding Ca2+. The adherence was remarkably inhibited by sugars containing D-galactose and n-acetyl-D-galactosamine. Treatment of neuraminidase-treated epithelial cells with sodium metaperiodate or alpha- and beta-galactosidase did not decrease the adherence. These data suggest that adherence of E. corrodens 1073 to human buccal epithelial cells may require the interaction of lectin-like proteins on the bacterial surface with galactose-like receptors on the surface of epithelial cells. PMID:6260661

  7. Ouabain modulates epithelial cell tight junction

    PubMed Central

    Larre, Isabel; Lazaro, Amparo; Contreras, Ruben G.; Balda, Maria S.; Matter, Karl; Flores-Maldonado, Catalina; Ponce, Arturo; Flores-Benitez, David; Rincon-Heredia, Ruth; Padilla-Benavides, Teresita; Castillo, Aída; Shoshani, Liora; Cereijido, Marcelino

    2010-01-01

    Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell–cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K+ pumping nor disturb the K+ balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (JDEX). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, JDEX, and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell–cell contacts. PMID:20534449

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

    PubMed

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

    2016-01-01

    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. PMID:27431614

  9. SOX2 functions as a molecular rheostat to control the growth, tumorigenicity and drug responses of pancreatic ductal adenocarcinoma cells

    PubMed Central

    Wuebben, Erin L.; Wilder, Phillip J.; Cox, Jesse L.; Grunkemeyer, James A.; Caffrey, Thomas; Hollingsworth, Michael A.; Rizzino, Angie

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly deadly malignancy. Expression of the stem cell transcription factor SOX2 increases during progression of PDAC. Knockdown of SOX2 in PDAC cell lines decreases growth in vitro; whereas, stable overexpression of SOX2 in one PDAC cell line reportedly increases growth in vitro. Here, we reexamined the role of SOX2 in PDAC cells, because inducible SOX2 overexpression in other tumor cell types inhibits growth. In this study, four PDAC cell lines were engineered for inducible overexpression of SOX2 or inducible knockdown of SOX2. Remarkably, inducible overexpression of SOX2 in PDAC cells inhibits growth in vitro and reduces tumorigenicity. Additionally, inducible knockdown of SOX2 in PDAC cells reduces growth in vitro and in vivo. Thus, growth and tumorigenicity of PDAC cells is highly dependent on the expression of optimal levels of SOX2 – a hallmark of molecular rheostats. We also determined that SOX2 alters the responses of PDAC cells to drugs used in PDAC clinical trials. Increasing SOX2 reduces growth inhibition mediated by MEK and AKT inhibitors; whereas knockdown of SOX2 further reduces growth when PDAC cells are treated with these inhibitors. Thus, targeting SOX2, or its mode of action, could improve the treatment of PDAC. PMID:27145457

  10. STUDIES ON AN EPITHELIAL (GLAND) CELL JUNCTION

    PubMed Central

    Loewenstein, Werner R.; Kanno, Yoshinobu

    1964-01-01

    Membrane permeability of an epithelial cell junction (Drosophila salivary gland) was examined with intracellular microelectrodes and with fluorescent tracers. In contrast to the non-junctional cell membrane surface, which has a low permeability to ions (10-4 mho/cm2), the junctional membrane surface is highly permeable. In fact, it introduces no substantial restriction to ion flow beyond that in the cytoplasm; the resistance through a chain of cells (150 Ω cm) is only slightly greater than in extruded cytoplasm (100 Ω cm). The diffusion resistance along the intercellular space to the exterior, on the other hand, is very high. Here, there exists an ion barrier of, at least, 104Ω cm2. As a result, small ions and fluorescein move rather freely from one cell to the next, but do not leak appreciably through the intercellular space to the exterior. The organ here, rather than the single cell, appears to be the unit of ion environment. The possible underlying structural aspects are discussed. PMID:14206423

  11. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

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

    PubMed Central

    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

  13. Association between ALDH1+/CD133+ stem-like cells and tumor angiogenesis in invasive ductal breast carcinoma

    PubMed Central

    LV, XINQUAN; WANG, YINGZI; SONG, YIMIN; PANG, XIA; LI, HUIXIANG

    2016-01-01

    The growth and metastasis of tumors is dependent on angiogenesis; however, the association between tumor stem cells (TSCs) and tumor angiogenesis remains to be elucidated. The present study aimed to investigate the expression of the TSC markers aldehyde dehydrogenase 1 (ALDH1) and cluster of differentiation 133 (CD133) in invasive ductal breast carcinoma, and identify their correlation with tumor angiogenesis. Stem-like cells from the breast tissue of 120 patients, who were diagnosed with invasive ductal breast carcinoma at The First Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan, China) between January 2009 and December 2010, were collected by surgical resection and analyzed using immunohistochemical double staining. The expression of the vascular markers CD34, CD105 and vascular endothelial growth factor (VEGF) were determined using single staining. Overall, 25.83% (31/120) of the specimens contained a large number of ALDH1+/CD133+ stem-like cells (ALDH1+/CD133+ tumor). ALDH1+/CD133+ expression is associated with microvessel density, VEGF-positive rate and estrogen receptor expression (P<0.05); however, ALDH1+/CD133+ expression was not associated with age, tumor diameter, lymph node metastasis, histological classification, progesterone receptor expression or human epidermal growth factor receptor 2 expression (P>0.05). The ALDH1+/CD133+ tumor phenotype and expression of VEGF were identified to be correlated in the present study (P=0.020). The present study revealed a close association between breast cancer TSC markers, including ALDH1 and CD133, and tumor angiogenesis. The results of the present study may provide a novel target and treatment strategy for future studies investigating tumor growth and metastasis. PMID:26998072

  14. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  15. Ductal barriers in mammary epithelium

    PubMed Central

    Owens, Mark B; Hill, Arnold DK; Hopkins, Ann M

    2013-01-01

    Tissue barriers play an integral role in the biology and pathobiology of mammary ductal epithelium. In normal breast physiology, tight and adherens junctions undergo dynamic changes in permeability in response to hormonal and other stimuli, while several of their proteins are directly involved in mammary tumorigenesis. This review describes first the structure of mammary ductal epithelial barriers and their role in normal mammary development, examining the cyclical changes in response to puberty, pregnancy, lactation and involution. It then examines the role of adherens and tight junctions and the participation of their constituent proteins in mammary tumorigenic functions such as migration, invasion and metastasis. Finally, it discusses the potential of these adhesion proteins as both prognostic biomarkers and potential therapeutic targets in breast cancer. PMID:24665412

  16. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow. PMID:26641038

  17. Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

    PubMed

    Le Guen, Ludovic; Marchal, Stéphane; Faure, Sandrine; de Santa Barbara, Pascal

    2015-10-01

    The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration. PMID:26126787

  18. Myoepithelial cells from pleomorphic adenoma are not influenced by tumor conditioned media from breast ductal adenocarcinoma and melanoma cells: An in vitro study.

    PubMed

    Martinez, Elizabeth Ferreira; Demasi, Ana Paula Dias; Napimoga, Marcelo Henrique; Silva, Carolina Amália Barcellos; Navarini, Natalia Festugatto; Araújo, Ney Soares; DE Araújo, Vera Cavalcanti

    2015-01-01

    Myoepithelial cells have been implicated in the regulation of the transition from in situ to invasive neoplasia in salivary gland tumors. Considering the importance of the microenvironment of the tumor, the present in vitro study therefore analyzed the morphological and phenotypic changes undergone by benign myoepithelial cells from pleomorphic adenoma (PA) stimulated by tumor-conditioned medium. The benign myoepithelial cells were obtained from PA and were cultured with fibronectin extracellular matrix protein, supplemented with tumor-conditioned medium, which was harvested from breast ductal adenocarcinoma AU-565 and melanoma Hs 852.T cells. The morphological alterations were assessed by immunofluorescence analysis using vimentin antibody. The α-smooth muscle actin (α-SMA) and fibroblast growth factor (FGF)-2 proteins were analyzed by indirect immunofluorescence and quantitative polymerase chain reaction (qPCR). No morphological changes were observed in the myoepithelial cells cultured in fibronectin protein under stimulation from either tumor-conditioned medium. The immunofluorescence results, which were supported by qPCR analysis, revealed that only α-SMA was upregulated in the fibronectin substratum, with or without tumor-conditioned medium obtained from breast ductal adenocarcinoma and melanoma cells. No significant difference in FGF-2 mRNA expression was detected when the cells were cultured either in the tumor-conditioned medium or in the fibronectin substratum. The tumor-conditioned medium harvested from breast ductal adenocarcinoma and melanoma did not affect myoepithelial cell differentiation and function, which was reflected by the fact that there was no observed increase in α-SMA and FGF-2 expression, respectively.

  19. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    PubMed

    Knodler, Leigh A; Nair, Vinod; Steele-Mortimer, Olivia

    2014-01-01

    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.

  20. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    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. PMID:25506353

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

    PubMed

    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. PMID:25506353

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

    PubMed

    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.

  3. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  4. Impaired oxidative phosphorylation regulates necroptosis in human lung epithelial cells.

    PubMed

    Koo, Michael Jakun; Rooney, Kristen T; Choi, Mary E; Ryter, Stefan W; Choi, Augustine M K; Moon, Jong-Seok

    2015-08-28

    Cellular metabolism can impact cell life or death outcomes. While metabolic dysfunction has been linked to cell death, the mechanisms by which metabolic dysfunction regulates the cell death mode called necroptosis remain unclear. Our study demonstrates that mitochondrial oxidative phosphorylation (OXPHOS) activates programmed necrotic cell death (necroptosis) in human lung epithelial cells. Inhibition of mitochondrial respiration and ATP synthesis induced the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and necroptotic cell death. Furthermore, we demonstrate that the activation of AMP-activated protein kinase (AMPK), resulting from impaired mitochondrial OXPHOS, regulates necroptotic cell death. These results suggest that impaired mitochondrial OXPHOS contributes to necroptosis in human lung epithelial cells.

  5. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

    This book examines the advances being made in the study of the physiology, cell biology, and molecular genetics of cystic fibrosis. Emphasis is placed on various areas of research that involve epithelial cells (e.g., the CF-specific phenotypes exhibited by epithelial cells, abnormalities in epithelium ion transport, chloride channel regulation in CF epithelial.) Coverage is presented on the current status of CF, including data on the incidence of the disease, its mode of inheritance, chromosomal localization, genetic heterogeneity, and screening and management.

  6. Growth of corneal epithelial cells over in situ therapeutic contact lens after simple limbal epithelial transplantation (SLET)

    PubMed Central

    Bhalekar, Swapnil; Sangwan, Virender S; Basu, Sayan

    2013-01-01

    An 11-year-old boy underwent simple limbal epithelial transplantation (SLET) from the healthy right eye to his left eye for total limbal stem cell deficiency. One month later, corneal surface epithelialised and whitish plaques overlying the transplants were seen inferiorly. Those plaques were adherent to the surface of the contact lens and underlying corneal surface had smooth elevations. Similar findings were noted in a 23-year man following cyanoacrylate glue application for corneal perforation. On histological and immunohistochemical analysis, cells lining the contact lenses were identified as corneal epithelial cells. These cases illustrate epithelial cell growth on the contact lens and epithelial hyperplasia on corresponding surface of the cornea. Exorbitant proliferation of the epithelial cells may be owing to young age; therefore, early contact lens removal after SLET in young age, can possibly avoid epithelial hyperplasia. This also reiterates the possibility of using contact lens as a scaffold to grow epithelial cells. PMID:23814196

  7. Alignment of cell division axes in directed epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

    2014-11-01

    Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

  8. Sustained co-cultivation with human placenta-derived MSCs enhances ALK5/Smad3 signaling in human breast epithelial cells, leading to EMT and differentiation.

    PubMed

    Yoo, Young A; Kang, Myoung Hee; Kim, Byung Soo; Kim, Jun Suk; Seo, Jae Hong

    2009-06-01

    The interaction between mammary epithelial cells and their surrounding microenvironment are important in the development of the mammary gland. Thus, mesenchymal stem cells (MSCs), which retain pluripotency for various mesenchymal lineages, may provide a permissive environment for the morphologic alteration and differentiation of mammary epithelial cells. To this end, we investigated whether the interactions between mammary epithelial cells and human placenta-derived MSCs (hPMSC) affect the morphology, proliferation, and differentiation of epithelial cells in a co-culture system. We show that after co-culture with hPMSCs, human mammary epithelial cell lines (MCF-10F and HEMC) underwent significant morphologic alterations and a dramatic increase in ductal-alveolar branching, which was accompanied by a decrease or loss of the epithelial marker E-cadherin and a gain of the mesenchymal markers, alpha-SMA and vimentin. MCF-10F and HEMC proliferation was also inhibited in the presence of hPMSCs, and this retardation in growth was due to cell cycle arrest. Furthermore, in MCF-10F and HMEC cells, hPMSCs induced the production of lipid droplets, milk fat globule protein, and milk protein lactoferrin, which are markers of functional mammary differentiation. We also noticed an elevation in ALK5 and phosphorylated Smad3 protein levels upon hPMSC co-culture. Strikingly, the changes in morphology, proliferation, and differentiation were reversed by treatment with ALK5 or Smad3 knockdown in MCF-10F/hPMSC co-cultures. Collectively, our findings suggest that co-cultivation with hPMSCs leads to epithelial to mesenchymal transition (EMT) and differentiation of human breast epithelial cells through the ALK5/Smad3 signaling pathway. PMID:19375841

  9. A novel function of colony-stimulating factor 1 receptor in hTERT immortalization of human epithelial cells.

    PubMed

    Li, N F; Kocher, H M; Salako, M A; Obermueller, E; Sandle, J; Balkwill, F

    2009-02-01

    The receptor for macrophage colony-stimulating factor 1 receptor (CSF1R) is a product of the proto-oncogene c-fms and a member of the class III transmembrane tyrosine kinase receptor family. Earlier, we described increased mRNA expression of CSF1R in human telomerase reverse transcriptase (hTERT) immortalized human ovarian surface epithelial (IOSE) cell lines derived from a single donor. Here, we further describe that CSF1R is upregulated at both the mRNA and protein level in hTERT immortalized human normal OSE cells from two different donors and in hTERT immortalized human pancreatic ductal epithelial cells. CSF1R was not upregulated in hTERT immortalized epithelial clones that subsequently underwent senescence or in immortalized fibroblasts. Upon stimulation by the CSF1R ligand CSF1, the immortalized epithelial cell lines showed rapid internalization of CSF1R with concomitant down-modulation and colocalization of phosphorylated NFkappaBp65 with hTERT protein, hTERT translocation into the nucleus and the binding of c-Myc to the hTERT promoter region. Reducing the expression of CSF1R using short hairpin interfering RNA abolished these effects and also decreased cell survival and the number of population doublings under suboptimal culture conditions. The telomerase inhibitor GRN163L confirmed a role for telomerase in the cleavage of the intracellular domain of CSF1R. On the basis of these findings, we suggest that CSF1R may be a critical factor facilitating hTERT immortalization of epithelial cells.

  10. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  11. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis.

  12. Phase I/II Study of IMMU-132 in Patients With Epithelial Cancers

    ClinicalTrials.gov

    2016-09-20

    Colorectal Cancer; Gastric Adenocarcinoma; Esophageal Cancer; Hepatocellular Carcinoma; Non-small Cell Lung Cancer; Small Cell Lung Cancer; Ovarian Epithelial Cancer; Carcinoma Breast Stage IV; Hormone-refractory Prostate Cancer; Pancreatic Ductal Adenocarcinoma; Head and Neck Cancers- Squamous Cell; Renal Cell Cancer; Urinary Bladder Neoplasms; Cervical Cancer; Endometrial Cancer; Follicular Thyroid Cancer; Glioblastoma Multiforme

  13. Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells

    PubMed Central

    Jimbo, Masaya; Blanco, Fernando F.; Screnci, Brad A.; Cosma, Gabriela L.; Alexeev, Vitali; Gonye, Gregory E.; Yeo, Charles J.; Sawicki, Janet A.; Winter, Jordan M.; Brody, Jonathan R.

    2015-01-01

    Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR’s role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA. PMID:26314962

  14. Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

    PubMed

    Jimbo, Masaya; Blanco, Fernando F; Huang, Yu-Hung; Telonis, Aristeidis G; Screnci, Brad A; Cosma, Gabriela L; Alexeev, Vitali; Gonye, Gregory E; Yeo, Charles J; Sawicki, Janet A; Winter, Jordan M; Brody, Jonathan R

    2015-09-29

    Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR's role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

  15. Crystal violet staining to quantify Candida adhesion to epithelial cells.

    PubMed

    Negri, M; Gonçalves, V; Silva, S; Henriques, M; Azeredo, J; Oliveira, R

    2010-01-01

    In vitro studies of adhesion capability are essential to characterise the virulence of Candida species. However, the assessment of adhesion by traditional methods is time-consuming. The aim of the present study is the development of a simple methodology using crystal violet staining to quantify in vitro adhesion of different Candida species to epithelial cells. The experiments are performed using Candida albicans (ATCC 90028), C. glabrata (ATCC 2001), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 750). A human urinary bladder epithelial cell line (TCC-SUP) is used. Yeast and epithelial cells were stained with crystal violet, epithelial cells were then destained using intermediate washing, and the dye in the yeast cells was extracted with acetic acid. The method was validated for the different Candida reference species by comparison with traditional microscope observation and enumeration. The method was then used to assess Candida adhesion to epithelial cells and also to silicone. For all Candida spp. high correlation values (r2= 0.9724-0.9997) between the number of adherent yeasts (microscope enumeration) and absorbance values were obtained for an inoculum concentration >10(6) cells/mL. The proposed technique was easy to perform and reproducible, enabling the determination of adhesion ability of Candida species to an epithelial cell line. PMID:20973406

  16. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

    Eule, Stephan; Metzger, Jakob; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Grosshans, Joerg; Wolf, Fred

    2015-03-01

    We found that the constriction of epithelial cell contacts during intercalation in germ band extension in Drosophila embryos follows intriguingly simple quantitative laws. The mean contact length < L > follows < L > (t) ~(T - t) α , where T is the finite collapse time; the time dependent variance of contact length is proportional to the square of the mean; finally the time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations suggest that the dynamics of contact collapse can be captured by a stochastic differential equation analytically tractable in small noise approximation. Here, we present such a model, providing an effective description of the non-equilibrium statistical mechanics of contact collapse. All model parameters are fixed by measurements of time dependent mean and variance of contact lengths. The model predicts the contact length covariance function that we obtain in closed form. The contact length covariance function closely matches experimental observations suggesting that the model well captures the dynamics of contact collapse.

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

    SciTech Connect

    Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot; Egan, Jim J.; Doran, Peter P.

    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.

  18. mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells

    PubMed Central

    2014-01-01

    Background Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist. Methods Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses. Results Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-β1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-β1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-β1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP. Conclusions Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs. PMID:24678619

  19. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line. PMID:20400167

  20. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

  1. Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model

    PubMed Central

    Juuti-Uusitalo, Kati M; Kaukinen, Katri; Mäki, Markku; Tuimala, Jarno; Kainulainen, Heikki

    2006-01-01

    Background The TGFβ1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFβ1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation. Results The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFβ1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes. Conclusion Our results reveal potential new signaling pathways and several new genes affected by TGFβ in epithelial cell differentiation. The differentiation induced by TGFβ1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells. PMID:17074098

  2. Influence of the mesenchymal cell source on oral epithelial development.

    PubMed

    Kinikoglu, Beste; Rovere, Marie Rose; Haftek, Marek; Hasirci, Vasif; Damour, Odile

    2012-03-01

    The extent of the influence of mesenchymal tissue on epithelial development is still debated, and elucidation of epithelial-mesenchymal interactions should be of relevance for controlling normal as well as pathological growth and development. The aim of the present study was to elucidate the influence of the mesenchymal cell type on oral mucosa epithelial development in vitro, using tissue-engineering principles, by including three different sources for mesenchymal cell type, viz. oral mucosa, skin and cornea, each of them presenting a distinct type of epithelium in situ. We investigated epithelial-mesenchymal interactions, considering both morphological criteria and protein expression (filaggrin, keratin 10, keratin 12, keratin 13 and laminin 5). The results of the histology, immunohistochemistry and transmission electron microscopy of the three types of tissue-engineered constructs composed of mesenchymal cells of different sources (oral, dermal and corneal fibroblasts) and of the same oral epithelial cells showed that the mesenchymal cell source had a significant influence on the thickness and ultrastructure of the epithelium, but not on the differentiation of oral epithelial cells, which might be an intrinsic property of these cells due to their genetic programming. PMID:21548135

  3. Isolation and characterization of cutaneous epithelial stem cells

    PubMed Central

    Jensen, Uffe B.; Ghazizadeh, Soosan; Owens, David M.

    2014-01-01

    SUMMARY During homeostasis, adult mammalian skin turnover is maintained by a number of multipotent and unipotent epithelial progenitors located either in the epidermis, hair follicle or sebaceous gland. Recent work has illustrated that these various progenitor populations reside in regionalized niches and are phenotypically distinct from one another. This degree of heterogeneity within the progenitor cell landscape in the cutaneous epithelium complicates our ability to target, purify and manipulate cutaneous epithelial stem cell subpopulations in adult skin. The techniques outlined in this chapter describe basic procedures for the isolation and purification of murine epithelial progenitors and assessing their capacity for ex vivo propagation. PMID:23483387

  4. High ROR2 expression in tumor cells and stroma is correlated with poor prognosis in pancreatic ductal adenocarcinoma

    PubMed Central

    Huang, Jianfei; Fan, Xiangjun; Wang, Xudong; Lu, Yuhua; Zhu, Huijun; Wang, Wei; Zhang, Shu; Wang, Zhiwei

    2015-01-01

    RTK-like orphan receptor 2 (ROR2) is overexpressed in several cancers and has tumorigenic activity. However, the expression of ROR2 and its functional and prognostic significance have yet to be evaluated in pancreatic ductal adenocarcinoma (PDAC). Quantitative real-time polymerase chain reaction was used to characterize the expression of ROR2 mRNA in PDAC, corresponding peritumoral tissues, and PDAC cell lines. Immunohistochemical analysis with tissue microarrays was used to evaluate ROR2 expression in PDAC and to investigate the relationship of this expression to clinicopathological factors and prognosis. The expression of ROR2 mRNA and protein was significantly higher in PDAC than in normal pancreatic tissues. High cytoplasmic ROR2 expression in cancer cells was significantly associated with a primary tumor, distant metastasis, and TNM stage, and high stromal ROR2 expression was significantly associated with regional lymph node metastasis and TNM stage. The Kaplan–Meier method and Cox regression analyses showed that high ROR2 expression in tumor cytoplasm or stromal cells was significantly associated with malignant attributes and reduced survival in PDAC. We present strong evidence that ROR2 could be used as an indicator of poor prognosis and could represent a novel therapeutic target for PDAC. PMID:26259918

  5. TSLP in Epithelial Cell and Dendritic Cell Cross Talk

    PubMed Central

    Liu, Yong-Jun

    2013-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells that have the ability to sense infection and tissue stress, sample and present antigen to T lymphocytes, and instruct the initiation of different forms of immunity and tolerance. The functional versatility of DCs depends on their remarkable ability to translate collectively the information from the invading microbes, as well as their resident tissue microenvironments. Recent progress in understanding Toll-like receptor (TLR) biology has illuminated the mechanisms by which DCs link innate and adaptive antimicrobial immune responses. However, how tissue microenvironments shape the function of DCs has remained elusive. Recent studies of TSLP (thymic stromal lymphopoietin), an epithelial cell-derived cytokine that strongly activates DCs, provide strong evidence at a molecular level that epithelial cells/tissue microenvironments directly communicate with DCs, the professional antigen-presenting cells of the immune system. We review recent progress on how TSLP expressed within thymus and peripheral lymphoid and nonlymphoid tissues regulates DC-mediated central tolerance, peripheral T cell homeostasis, and inflammatory Th2 responses. PMID:19231591

  6. Changes in the Submandibular Salivary Gland Epithelial Cell Subpopulations During Progression of Sjögren's Syndrome-Like Disease in the NOD/ShiLtJ Mouse Model.

    PubMed

    Gervais, Elise M; Desantis, Kara A; Pagendarm, Nicholas; Nelson, Deirdre A; Enger, Tone; Skarstein, Kathrine; Liaaen Jensen, Janicke; Larsen, Melinda

    2015-09-01

    Sjögren's syndrome (SS), an autoimmune exocrinopathy, is associated with dysfunction of the secretory salivary gland epithelium, leading to xerostomia. The etiology of SS disease progression is poorly understood as it is typically not diagnosed until late stage. Since mouse models allow the study of disease progression, we investigated the NOD/ShiLtJ mouse to explore temporal changes to the salivary epithelium. In the NOD/ShiLtJ model, SS presents secondary to autoimmune diabetes, and SS disease is reportedly fully established by 20 weeks. We compared epithelial morphology in the submandibular salivary glands (SMG) of NOD/ShiLtJ mice with SMGs from the parental strain at 12, 18, and 22 weeks of age and used immunofluorescence to detect epithelial proteins, including the acinar marker, aquaporin 5, ductal cell marker, cytokeratin 7, myoepithelial cell marker, smooth muscle α-actin, and the basal cell marker, cytokeratin 5, while confirming immune infiltrates with CD45R. We also compared these proteins in the labial salivary glands of human SS patients with control tissues. In the NOD/ShiLtJ SMG, regions of lymphocytic infiltrates were not associated with widespread epithelial tissue degradation; however, there was a decrease in the area of the gland occupied by secretory epithelial cells in favor of ductal epithelial cells. We observed an expansion of cells expressing cytokeratin 5 within the ducts and within the smooth muscle α-actin(+) basal myoepithelial population. The altered acinar/ductal ratio within the NOD/ShiLtJ SMG likely contributes to salivary hypofunction, while the expansion of cytokeratin 5 positive-basal cells may reflect loss of function or indicate a regenerative response.

  7. Fibronectin Expression Modulates Mammary Epithelial Cell Proliferation during Acinar Differentiation

    PubMed Central

    Williams, Courtney M.; Engler, Adam J.; Slone, R. Daniel; Galante, Leontine L.; Schwarzbauer, Jean E.

    2009-01-01

    The mammary gland consists of a polarized epithelium surrounded by a basement membrane matrix that forms a series of branching ducts ending in hollow, sphere-like acini. Essential roles for the epithelial basement membrane during acinar differentiation, in particular laminin and its integrin receptors, have been identified using mammary epithelial cells cultured on a reconstituted basement membrane. Contributions from fibronectin, which is abundant in the mammary gland during development and tumorigenesis, have not been fully examined. Here, we show that fibronectin expression by mammary epithelial cells is dynamically regulated during the morphogenic process. Experiments with synthetic polyacrylamide gel substrates implicate both specific extracellular matrix components, including fibronectin itself, and matrix rigidity in this regulation. Alterations in fibronectin levels perturbed acinar organization. During acinar development, increased fibronectin levels resulted in overproliferation of mammary epithelial cells and increased acinar size. Addition of fibronectin to differentiated acini stimulated proliferation and reversed growth arrest of mammary epithelial cells negatively affecting maintenance of proper acinar morphology. These results show that expression of fibronectin creates a permissive environment for cell growth that antagonizes the differentiation signals from the basement membrane. These effects suggest a link between fibronectin expression and epithelial cell growth during development and oncogenesis in the mammary gland. PMID:18451144

  8. Biliary epithelial cells proliferate during oxygenated ex situ liver culture

    PubMed Central

    Bian, Congwen; Du, Yiqi; Ding, Rui; Huang, Jun; Dai, Yan; Bao, Sujin; Zhao, Lijuan; Shen, Hefang; Dong, Jing; Xu, Jianjian; Xiong, Qiru; Xu, Lili

    2016-01-01

    Biliary complications remain a major source of morbidity in liver transplant patients. Among these complications, nonanastomotic biliary strictures (NAS) are especially common and they are frequently therapy resistant in part because biliary epithelial cells are more sensitive to warm ischemic injury than hepatocytes. It has been a challenge to maintain the physiological function of biliary epithelial cells during liver transplantation. In this work, we have examined the effect of oxygen on proliferation of biliary epithelial cells in the rat livers obtained from donation after circulatory death (DCD). Twelve rat livers from DCD were divided into two groups. Livers in the control group were isolated following a standard procedure without oxygen supply. Livers in the experimental group were isolated with a constant supply of oxygen. All livers were then connected to an ex situ liver culture system in the presence of bromodeoxyuridine (BrdU), a thymidine analogue and a marker for cell proliferation. After 6 hours of normothermic ex situ liver culture, morphology and DNA replication in hepatocytes and biliary epithelial cells were assessed and compared between the two groups. We found that about 4.5% of the biliary epithelial cells in the experimental group proliferated compared with only 0.4% of cells in the control based on BrdU staining. No significant change in cell morphology was observed in those cells between the two groups. Thus, our results indicate that oxygen supply is required for maintenance of the physiological function of biliary epithelial cells during liver transplant and suggest that a constant oxygen supply during liver isolation along with ex situ liver organ culture can enhance the repair of biliary epithelial cell injury during liver transplantation. PMID:27725875

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

    SciTech Connect

    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.

  10. Inhibition of corneal epithelial cell migration by cadmium and mercury

    SciTech Connect

    Ubels, J.L.; Osgood, T.B. Medical Coll. of Wisconsin, Milwaukee )

    1991-02-01

    In a previous comparative study of corneal healing in fish, the authors observed that corneal epithelial healing occurs very rapidly in vivo in the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) with a 6-mm diameter wound on the mammalian cornea. This rapid healing which permits prompt restoration of the epithelial barrier is apparently an adaptation to the large ionic and osmotic gradients between the environment and the intraocular fluids of the fish. These observations suggested that epithelial healing in the sculpin cornea might be useful model in aquatic biomedical toxicology if an in vitro method for measurement of healing rates could be developed. In this report the authors demonstrate that sculpin eyes maintained in short-term organ culture have a rapid corneal epithelial healing response and that this model can be used to demonstrate the toxic effects of heavy metals on epithelial cell migration.

  11. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    SciTech Connect

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with {sup 3}H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-{beta} did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 {times} 10{sup 6} sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined.

  12. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-12-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction.

  13. Selective inhibition of pancreatic ductal adenocarcinoma cell growth by the mitotic MPS1 kinase inhibitor NMS-P715.

    PubMed

    Slee, Roger B; Grimes, Brenda R; Bansal, Ruchi; Gore, Jesse; Blackburn, Corinne; Brown, Lyndsey; Gasaway, Rachel; Jeong, Jaesik; Victorino, Jose; March, Keith L; Colombo, Riccardo; Herbert, Brittney-Shea; Korc, Murray

    2014-02-01

    Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy.

  14. Role of autophagy in the regulation of epithelial cell junctions.

    PubMed

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  15. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. PMID:20113446

  16. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing.

  17. Inflammatory infiltrate in invasive lobular and ductal carcinoma of the breast.

    PubMed Central

    Lee, A. H.; Happerfield, L. C.; Millis, R. R.; Bobrow, L. G.

    1996-01-01

    The significance of inflammation in carcinoma of the breast is controversial. Little attention has been paid to different patterns of inflammation or inflammation associated with different histological types of carcinoma. We have looked at the pattern of inflammation in 123 invasive mammary carcinomas (including 46 lobular), and characterised the inflammatory cells with immunohistochemistry in 21. We found different patterns of inflammation in ductal and lobular carcinoma. Diffuse inflammation was seen more in ductal carcinoma, particularly of high grade, and was predominantly composed of macrophages and T cells. It was associated with necrosis, but the correlation was weak, suggesting that other factors are important. Perilobular inflammation was seen most frequently in lobular and high-grade ductal carcinomas, particularly at the tumour edge. Perivascular inflammation was also largely at the tumour edge, but was not more common in any tumour type. In contrast to the diffuse inflammation, the perivascular and perilobular inflammation was composed of T and B cells. Normal lobules at the tumour edge showed consistent expression of HLA-DR, whereas lobules away from the tumour were negative. A combination of perilobular and perivascular inflammation composed of B and T cells with epithelial expression of HLA-DR mimicking lymphocytic lobulitis was seen more frequently in lobular than ductal carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:8795584

  18. DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

    PubMed

    Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

    2014-04-01

    DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

  19. Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ

    PubMed Central

    Whitaker-Menezes, Diana; Martinez-Outschoorn, Ubaldo E; Flomenberg, Neal; Birbe, Ruth C; Witkiewicz, Agnieszka K; Howell, Anthony; Pavlides, Stephanos; Tsirigos, Aristotelis; Ertel, Adam; Pestell, Richard G; Broda, Paolo; Minetti, Carlo

    2011-01-01

    We have recently proposed a new mechanism for explaining energy transfer in cancer metabolism. In this scenario, cancer cells behave as metabolic parasites, by extracting nutrients from normal host cells, such as fibroblasts, via the secretion of hydrogen peroxide as the initial trigger. Oxidative stress in the tumor microenvironment then leads to autophagy-driven catabolism, mitochondrial dys-function and aerobic glycolysis. This, in turn, produces high-energy nutrients (such as L-lactate, ketones and glutamine) that drive the anabolic growth of tumor cells, via oxidative mitochondrial metabolism. A logical prediction of this new “parasitic” cancer model is that tumor-associated fibroblasts should show evidence of mitochondrial dys-function (mitophagy and aerobic glycolysis). In contrast, epithelial cancer cells should increase their oxidative mitochondrial capacity. To further test this hypothesis, here we subjected frozen sections from human breast tumors to a staining procedure that only detects functional mitochondria. This method detects the in situ enzymatic activity of cytochrome C oxidase (COX), also known as Complex IV. Remarkably, cancer cells show an over-abundance of COX activity, while adjacent stromal cells remain essentially negative. Adjacent normal ductal epithelial cells also show little or no COX activity, relative to epithelial cancer cells. Thus, oxidative mitochondrial activity is selectively amplified in cancer cells. Although COX activity staining has never been applied to cancer tissues, it could now be used routinely to distinguish cancer cells from normal cells, and to establish negative margins during cancer surgery. Similar results were obtained with NADH activity staining, which measures Complex I activity, and succinate dehydrogenase (SDH) activity staining, which measures Complex II activity. COX and NADH activities were blocked by electron transport inhibitors, such as Metformin. This has mechanistic and clinical implications

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

    PubMed Central

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

    2015-01-01

    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. PMID:25977510

  1. Trefoil peptides promote restitution of wounded corneal epithelial cells.

    PubMed

    Göke, M N; Cook, J R; Kunert, K S; Fini, M E; Gipson, I K; Podolsky, D K

    2001-04-01

    The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

  2. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    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.

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

    PubMed

    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. PMID:26414404

  4. Lingual Epithelial Stem Cells and Organoid Culture of Them.

    PubMed

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-28

    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.

  5. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  6. Development of human epithelial cell systems for radiation risk assessment.

    PubMed

    Yang, C H; Craise, L M

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells. PMID:11538024

  7. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  8. Successful Salvage Chemotherapy with FOLFIRINOX for Recurrent Mixed Acinar Cell Carcinoma and Ductal Adenocarcinoma of the Pancreas in an Adolescent Patient

    PubMed Central

    Pfrommer, Sarah; Weber, Achim; Dutkowski, Philipp; Schäfer, Niklaus G.; Müllhaupt, Beat; Bourquin, Jean-Pierre; Breitenstein, Stefan; Pestalozzi, Bernhard C.; Stenner, Frank; Renner, Christoph; D'Addario, Giannicola; Graf, Hans-Jörg; Knuth, Alexander; Clavien, Pierre-Alain; Samaras, Panagiotis

    2013-01-01

    Pancreatic tumors are rare in children and adolescents. Here, we report the case of a 15-year-old boy who presented with a mixed acinar cell carcinoma/ductal adenocarcinoma with blastomatous components. He received multimodal treatment including various chemotherapy regimens and multistep surgery including liver transplantation. Introduction of FOLFIRINOX after relapse repeatedly achieved a durable metabolic and clinical response with good quality of life. PMID:24163668

  9. Successful Salvage Chemotherapy with FOLFIRINOX for Recurrent Mixed Acinar Cell Carcinoma and Ductal Adenocarcinoma of the Pancreas in an Adolescent Patient.

    PubMed

    Pfrommer, Sarah; Weber, Achim; Dutkowski, Philipp; Schäfer, Niklaus G; Müllhaupt, Beat; Bourquin, Jean-Pierre; Breitenstein, Stefan; Pestalozzi, Bernhard C; Stenner, Frank; Renner, Christoph; D'Addario, Giannicola; Graf, Hans-Jörg; Knuth, Alexander; Clavien, Pierre-Alain; Samaras, Panagiotis

    2013-01-01

    Pancreatic tumors are rare in children and adolescents. Here, we report the case of a 15-year-old boy who presented with a mixed acinar cell carcinoma/ductal adenocarcinoma with blastomatous components. He received multimodal treatment including various chemotherapy regimens and multistep surgery including liver transplantation. Introduction of FOLFIRINOX after relapse repeatedly achieved a durable metabolic and clinical response with good quality of life. PMID:24163668

  10. Autophagic and apoptotic cell death in amniotic epithelial cells.

    PubMed

    Shen, Z-Y; Li, E-M; Lu, S-Q; Shen, J; Cai, Y-M; Wu, Y-E; Zheng, R-M; Tan, L-J; Xu, L-Y

    2008-11-01

    The aim of this paper is to determine if autophagic cell death is associated with apoptosis and whether it participates in the process of term amniotic rupture. Forty pieces of fresh term amnions, including twenty from a position near the margin of the placentas and twenty from the margin of the naturally ruptured part of the placentas in term gestation were collected, respectively. The amnions were examined by scanning electron microscopy (SEM) and amniotic epithelial (AE) cells were examined by transmission electron microscopy (TEM). Autophagic and apoptotic cell death (PCD) were assayed by laser scanning confocal microscopy (LSCM) or flow cytometry using monodansylcadaverin (MDC) and propidium iodide (PI) stain. BCL(2) and BAX were examined by immunoblotting. Under SEM the amniotic epithelia appeared normal in the position near the placenta. They had an atrophied appearance in the margin of their natural broken parts. In the AE cells PCD was divided into three subtypes by TEM: autophagic cell death with positive stains of MDC and PI; apoptotic cell death; and the mixed type. Quantitative detection showed that there were more death cells, including autophagic and apoptotic, in the AE cells near the ruptured parts than near the placentas. An increased expression of BAX and a decreased expression of BCL(2) protein in the AE cells near the broken margin were observed. Apoptotic and autophagic cell death by the intrinsic pathway are the basic event in the AE cell and they are involved in the cause of membrane rupture of the human amnion in term gestation.

  11. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.15034.001 PMID:27431614

  13. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  14. Trans-sialidase Stimulates Eat Me Response from Epithelial Cells

    PubMed Central

    Butler, Claire E; de Carvalho, Tecia M U; Grisard, Edmundo C; Field, Robert A; Tyler, Kevin M

    2013-01-01

    Epithelial cell invasion by the protozoan parasite Trypanosoma cruzi is enhanced by the presence of an enzyme expressed on its cell surface during the trypomastigote life cycle stage. The enzyme, trans-sialidase (TS), is a member of one of the largest gene families expressed by the parasite and the role of its activity in mediating epithelial cell entry has not hitherto been understood. Here we show that the T. cruzi TS generates an eat me signal which is capable of enabling epithelial cell entry. We have utilized purified, recombinant, active (TcTS) and inactive (TcTS2V0) TS coated onto beads to challenge an epithelial cell line. We find that TS activity acts upon G protein coupled receptors present at the epithelial cell synapse with the coated bead, thereby enhancing cell entry. By so doing, we provide evidence that TS proteins bind glycans, mediate the formation of distinct synaptic domains and promote macropinocytotic uptake of microparticles into a perinuclear compartment in a manner which may emulate entosis. PMID:23601193

  15. Mechanobiology in Lung Epithelial Cells: Measurements, Perturbations, and Responses

    PubMed Central

    Waters, Christopher M.; Roan, Esra; Navajas, Daniel

    2015-01-01

    Epithelial cells of the lung are located at the interface between the environment and the organism and serve many important functions including barrier protection, fluid balance, clearance of particulate, initiation of immune responses, mucus and surfactant production, and repair following injury. Because of the complex structure of the lung and its cyclic deformation during the respiratory cycle, epithelial cells are exposed to continuously varying levels of mechanical stresses. While normal lung function is maintained under these conditions, changes in mechanical stresses can have profound effects on the function of epithelial cells and therefore the function of the organ. In this review, we will describe the types of stresses and strains in the lungs, how these are transmitted, and how these may vary in human disease or animal models. Many approaches have been developed to better understand how cells sense and respond to mechanical stresses, and we will discuss these approaches and how they have been used to study lung epithelial cells in culture. Understanding how cells sense and respond to changes in mechanical stresses will contribute to our understanding of the role of lung epithelial cells during normal function and development and how their function may change in diseases such as acute lung injury, asthma, emphysema, and fibrosis. PMID:23728969

  16. Ozone exposed epithelial cells modify cocultured natural killer cells

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.

    2013-01-01

    Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs. PMID:23241529

  17. Cell division and the maintenance of epithelial order

    PubMed Central

    Ragkousi, Katerina

    2014-01-01

    Epithelia are polarized layers of adherent cells that are the building blocks for organ and appendage structures throughout animals. To preserve tissue architecture and barrier function during both homeostasis and rapid growth, individual epithelial cells divide in a highly constrained manner. Building on decades of research focused on single cells, recent work is probing the mechanisms by which the dynamic process of mitosis is reconciled with the global maintenance of epithelial order during development. These studies reveal how symmetrically dividing cells both exploit and conform to tissue organization to orient their mitotic spindles during division and establish new adhesive junctions during cytokinesis. PMID:25349258

  18. Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.

    PubMed

    Nakahashi-Oda, Chigusa; Udayanga, Kankanam Gamage Sanath; Nakamura, Yoshiyuki; Nakazawa, Yuta; Totsuka, Naoya; Miki, Haruka; Iino, Shuichi; Tahara-Hanaoka, Satoko; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira

    2016-04-01

    Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1(+)CD103(-)CD11b(+) dendritic cells (DCs) to produce interferon-β (IFN-β), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-β production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis. PMID:26855029

  19. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.

    PubMed

    Huang, Ling; Holtzinger, Audrey; Jagan, Ishaan; BeGora, Michael; Lohse, Ines; Ngai, Nicholas; Nostro, Cristina; Wang, Rennian; Muthuswamy, Lakshmi B; Crawford, Howard C; Arrowsmith, Cheryl; Kalloger, Steve E; Renouf, Daniel J; Connor, Ashton A; Cleary, Sean; Schaeffer, David F; Roehrl, Michael; Tsao, Ming-Sound; Gallinger, Steven; Keller, Gordon; Muthuswamy, Senthil K

    2015-11-01

    There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53(R175H) induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

  20. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids

    PubMed Central

    Huang, Ling; Holtzinger, Audrey; Jagan, Ishaan; BeGora, Michael; Lohse, Ines; Ngai, Nicholas; Nostro, Cristina; Wang, Rennian; Muthuswamy, Lakshmi B.; Crawford, Howard C.; Arrowsmith, Cheryl; Kalloger, Steve E.; Renouf, Daniel J.; Connor, Ashton A; Cleary, Sean; Schaeffer, David F.; Roehrl, Michael; Tsao, Ming-Sound; Gallinger, Steven; Keller, Gordon; Muthuswamy, Senthil K.

    2016-01-01

    There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells (PSCs) into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53R175H induced cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. Culture conditions are also defined for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture, phenotypic heterogeneity of the primary tumor, and retain patient-specific physiologic changes including hypoxia, oxygen consumption, epigenetic marks, and differential sensitivity to EZH2 inhibition. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies. PMID:26501191

  1. Angiomyolipoma with epithelial cysts: mimic of renal cell carcinoma.

    PubMed

    Rosenkrantz, Andrew B; Hecht, Elizabeth M; Taneja, Samir S; Melamed, Jonathan

    2010-01-01

    Angiomyolipoma with epithelial cysts (AMLEC) is a rare variant of angiomyolipoma with minimal fat that contains epithelial-lined cysts and may mimic a cystic renal cell carcinoma. While 17 cases have been described in the pathology literature since this entity was first described in 2006, the radiologic appearance was not demonstrated in any of these cases. We report the CT and MRI appearance of AMLEC found incidentally in a patient with lupus nephritis.

  2. Change in Cell Shape Is Required for Matrix Metalloproteinase-Induced Epithelial-Mesenchymal Transition of Mammary Epithelial Cells

    PubMed Central

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

    2010-01-01

    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-β-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. PMID:18506791

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

    SciTech Connect

    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.

  4. Primary human bronchial epithelial cells grown from explants.

    PubMed

    Yaghi, Asma; Zaman, Aisha; Dolovich, Myrna

    2010-01-01

    Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and < or =1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm(3) pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 microg/ml), fibronectin (10 microg/ml), and BSA (10 microg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37 degrees C in 5% CO(2) humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly

  5. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    PubMed Central

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  6. Inverted ductal papilloma of minor salivary glands.

    PubMed

    Hegarty, D J; Hopper, C; Speight, P M

    1994-08-01

    Inverted ductal papillomas are rare tumours of minor salivary glands. A case is reported on the lower lip of a 50-year-old man. The tumour showed a characteristic endophytic growth pattern and was composed of bulbous papillary projections of basaloid cells dilating and filling the superficial portion of the excretory duct. Histologically, inverted ductal papillomas resemble the inverted papilloma of the nose and paranasal sinuses, but are completely benign and are not associated with malignant change.

  7. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells.

    PubMed Central

    Paris, S; Boisvieux-Ulrich, E; Crestani, B; Houcine, O; Taramelli, D; Lombardi, L; Latgé, J P

    1997-01-01

    The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells. PMID:9119494

  8. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  9. Epithelial Stem Cells and Implications for Wound Repair

    PubMed Central

    Plikus, Maksim V.; Gay, Denise L.; Treffeisen, Elsa; Wang, Anne; Supapannachart, Rarinthip June; Cotsarelis, George

    2012-01-01

    Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis hasa mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new inter-follicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover cellular and signaling basis of this remarkable adult wound regeneration phenomenon. PMID:23085626

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

    PubMed Central

    St Johnston, Daniel

    2016-01-01

    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 epithelium1,2. Here we test this assumption in three types of Drosophila epithelia; 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 of 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 appears to be driven by lateral adhesion, which pulls cells born outside the epithelia layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  11. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  12. Characterization of discrete equine intestinal epithelial cell lineages

    PubMed Central

    Gonzalez, Liara M.; Kinnin, Leslie A.; Blikslager, Anthony T.

    2015-01-01

    OBJECTIVE To characterize epithelial cells of the small intestine and colon in horses without clinical gastrointestinal abnormalities with an emphasis on the stem cell niche constituents. SAMPLE Mucosal biopsy specimens from small and large intestines obtained from 12 horses euthanized for reasons unrelated to gastrointestinal disease or systemic disease. PROCEDURES Intestinal biopsy specimens were collected by sharp dissection immediately following euthanasia. Specimens were prepared for immunohistochemical, immunofluorescence, and transmission electron microscopic imaging to detect and characterize each epithelial cell type. Antibodies against protein biomarkers for cellular identification were selected on the basis of expression in other mammalian species. RESULTS Intestinal epithelial cell types were identified by means of immunostaining and morphological characterization with transmission electron microscopy. Some differences in biomarker expression and antibody cross-reactivity were identified in equine tissue, compared with other species. However, each known type of mucosal epithelial cell was identified in equine tissue. CONCLUSIONS AND CLINICAL RELEVANCE The methodology used can enhance detection of stem cells and progenitor cells as well as postmitotic cell lineages in equine intestinal tissues. Results may have relevance to regenerative potential of intestinal mucosa and survival in horses with colic. PMID:25815577

  13. Nipah Virus Entry and Egress from Polarized Epithelial Cells

    PubMed Central

    Lamp, Boris; Dietzel, Erik; Kolesnikova, Larissa; Sauerhering, Lucie; Erbar, Stephanie; Weingartl, Hana

    2013-01-01

    Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal. PMID:23283941

  14. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells. PMID:17460896

  15. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

  16. Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

    PubMed Central

    West, John D; Dorà, Natalie J; Collinson, J Martin

    2015-01-01

    In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed

  17. Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

    PubMed Central

    Duncan, M J; Emory, S A; Almira, E C

    1996-01-01

    Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed. PMID:8751909

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

    PubMed Central

    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

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

    PubMed

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

    2014-10-01

    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.

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

    PubMed Central

    Aoshiba, K; Nagai, A

    2003-01-01

    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. PMID:19570263

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

    PubMed Central

    Aoshiba, K; Nagai, A

    2003-01-01

    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.

  2. Overexpression of c-myc induces epithelial mesenchymal transition in mammary epithelial cells.

    PubMed

    Cho, Kyoung Bin; Cho, Min Kyong; Lee, Won Young; Kang, Keon Wook

    2010-07-28

    The c-myc gene is frequently overexpressed in human breast cancer and its target genes are involved in tumorigenesis. Epithelial mesenchymal transitions (EMT), where cells undergo a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype, are associated with invasion and motility of cancer cells. Basal E-cadherin expression was down-regulated in c-myc overexpressing MCF10A (c-myc-MCF10A) cells compared to GFP-overexpressing MCF10A (GFP-MCF10A) cells, while N-cadherin was distinctly increased in c-myc-MCF10A cells. Given that glycogen synthase kinase-3beta (GSK-3beta) and the snail axis have key roles in E-cadherin deregulation during EMT, we investigated the role of GSK-3beta/snail signaling pathways in the induction of EMT by c-myc overexpression. In contrast to GFP-MCF10A cells, both the transcriptional activity and the ubiquitination-dependent protein stability of snail were enhanced in c-myc-MCF10A cells, and this was reversed by GSK-3beta overexpression. We also found that c-myc overexpression inhibits GSK-3beta activity through activation of extracellular signal-regulated kinase (ERK). Inhibition of ERK by dominant negative mutant transfection or chemical inhibitor significantly suppressed snail gene transcription. These results suggest that c-myc overexpression during transformation of mammary epithelial cells (MEC) is involved in EMTs via ERK-dependent GSK-3beta inactivation and subsequent snail activation.

  3. The Crosstalk between Nrf2 and TGF-β1 in the Epithelial-Mesenchymal Transition of Pancreatic Duct Epithelial Cells.

    PubMed

    Arfmann-Knübel, Sarah; Struck, Birte; Genrich, Geeske; Helm, Ole; Sipos, Bence; Sebens, Susanne; Schäfer, Heiner

    2015-01-01

    Nrf2 and TGF-β1 both affect tumorigenesis in a dual fashion, either by preventing carcinogen induced carcinogenesis and suppressing tumor growth, respectively, or by conferring cytoprotection and invasiveness to tumor cells during malignant transformation. Given the involvement of Nrf2 and TGF-β1 in the adaptation of epithelial cells to persistent inflammatory stress, e.g. of the pancreatic duct epithelium during chronic pancreatitis, a crosstalk between Nrf2 and TGF-β1 can be envisaged. By using premalignant human pancreatic duct cells (HPDE) and the pancreatic ductal adenocarcinoma cell line Colo357, we could show that Nrf2 and TGF-β1 independently but additively conferred an invasive phenotype to HPDE cells, whereas acting synergistically in Colo357 cells. This was accompanied by differential regulation of EMT markers like vimentin, Slug, L1CAM and E-cadherin. Nrf2 activation suppressed E-cadherin expression through an as yet unidentified ARE related site in the E-cadherin promoter, attenuated TGF-β1 induced Smad2/3-activity and enhanced JNK-signaling. In Colo357 cells, TGF-β1 itself was capable of inducing Nrf2 whereas in HPDE cells TGF-β1 per-se did not affect Nrf2 activity, but enhanced Nrf2 induction by tBHQ. In Colo357, but not in HPDE cells, the effects of TGF-β1 on invasion were sensitive to Nrf2 knock-down. In both cell lines, E-cadherin re-expression inhibited the proinvasive effect of Nrf2. Thus, the increased invasion of both cell lines relates to the Nrf2-dependent downregulation of E-cadherin expression. In line, immunohistochemistry analysis of human pancreatic intraepithelial neoplasias in pancreatic tissues from chronic pancreatitis patients revealed strong Nrf2 activity already in premalignant epithelial duct cells, accompanied by partial loss of E-cadherin expression. Our findings indicate that Nrf2 and TGF-β1 both contribute to malignant transformation through distinct EMT related mechanisms accounting for an invasive phenotype

  4. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

  5. Sheep stromal-epithelial cell interactions and ovarian tumor progression.

    PubMed

    Wang-Johanning, Feng; Huang, Miao; Liu, Jinsong; Rycaj, Kiera; Plummer, Joshua B; Barnhart, Kirstin F; Satterfield, William C; Johanning, Gary L

    2007-11-15

    Previous studies suggest that underlying ovarian stromal cues may regulate the ovarian surface epithelium. However, little is known about the interaction between ovarian stromal cells (OSC) and ovarian surface epithelial cells (OSE) under normal physiologic and pathologic conditions, largely because of the lack of a suitable model. In the current study, the OSC obtained from a sheep were immortalized with SV-40 T/t antigen (designated IOSC) and telomerase reverse transcriptase (designated IOSCH), followed by transfection with the oncogenic allele of the human H-Ras oncogene (designated IOSChR). IOSC cells transfected with H-Ras before immortalization with telomerase were designated IOSCRH. These sheep OSCs were used in both in vitro and in vivo model systems to evaluate mechanisms by which OSCs influence ovarian tumor progression. Normal sheep OSCs were found to inhibit the growth of SKOV3 and OVCAR3 human ovarian cancer cells, but not normal sheep OSE and human OSE cells (hOSE137 cells). In contrast, IOSChR and IOSCRH cells stimulated the growth of normal sheep and human OSE cells, as well as cancer cells. These findings were confirmed by in vivo studies. Our data provide compelling support for the importance of stromal-epithelial cell interactions during tumor progression, and show for the first time that immortalized and transformed OSCs promote growth of ovarian epithelial tumors.

  6. [Methotrexate as inducer of proinflammatory cytokines by epithelial cells].

    PubMed

    Morón-Medina, Alejandra; Viera, Ninoska; de Morales, Thaís Rojas; Alcocer, Sirley; Bohorquez, Dinorath

    2014-03-01

    Methotrexate (MTX), a drug commonly used in childhood cancer, has also been indicated as a cytotoxic agent of the oral mucosa, which can trigger the inflammatory process and increase the vascularity of epithelial tissues during the early stages of oral mucositis. The aim of this study was to determine the production of proinflammatory cytokines IL-1beta, IL-6 y TNF-alpha in epithelial cell cultures treated with MTX. Epithelial cells of human larynx, obtained from the cell line Hep-2, were cultured with different doses of MTX during different incubation times. The drug cytotoxicity was analyzed by means of the colorimetric test, which is based on the metabolic reduction of the bromide of 3-(4, 5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT); and the proinflammatory cytokines production by the test enzyme-linked immunosorbent assay (ELISA). Cultures of HEp-2 cells showed increased production of proinflammatory cytokines at 72 hours with 0.32 microM of MTX. These results suggest that depending on the dose and exposure time, MTX alters the physiology of human epithelial cells, which may play an important role during the phases of initiation and development of oral mucositis. PMID:24758098

  7. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

  8. Establishment of a rat nasal epithelial tumor cell line.

    PubMed

    Hood, A T; Currie, D; Garte, S J

    1987-04-01

    A new cell line designated NAS 2BL has been established from a rat nasal tumor induced by inhalation of the direct-acting carcinogen methylmethane sulfonate. The cells are epithelial in morphology, have a generation time of 34 h, require 10% fetal bovine serum for optimal growth, and exhibit keratinization at confluence. The karyotype is aneuploid, with several marker chromosomes, and the cells are transformed by the criterion of nude mouse tumorigenicity.

  9. Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

    PubMed

    Panneck, Alexandra Regina; Rafiq, Amir; Schütz, Burkhard; Soultanova, Aichurek; Deckmann, Klaus; Chubanov, Vladimir; Gudermann, Thomas; Weihe, Eberhard; Krasteva-Christ, Gabriela; Grau, Veronika; del Rey, Adriana; Kummer, Wolfgang

    2014-12-01

    Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

  10. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    PubMed

    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.

  11. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    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. PMID:24065923

  12. Lung epithelial cell death induced by oil-dispersant mixtures.

    PubMed

    Wang, He; Shi, Yongli; Major, Danielle; Yang, Zhanjun

    2012-08-01

    The dispersants used in oil spill disasters are claimed to be safe, but increased solubility of high-molecular-weight components in crude oil is of public health concern. The water-accommodated fractions (WAF) of crude oil mixed with dispersants may become airborne and cause lung epithelial damage when inhaled. This study was designed to examine the cell death and related death pathways of lung epithelial cells in response to WAF. Cultured A549 cells were treated for 2 or 24h with different concentrations of WAF. The WAF was prepared by mixing each of the dispersants (Corexit EC9527A, Corexit EC9500A and Corexit EC9580A) with crude oil for extraction with PBS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay, lactate dehydrogenase assay, morphology and cleaved caspase 9 protein, and microtubule-associated protein 1 light chain 3 were all used to measure cell viability, necrosis, apoptosis and autophagy quantitation, respectively. Results showed that the WAF of oil-dispersant mixtures caused cell death in the lung epithelial cells, in a dose-dependent manner, with the major cellular pathways of necrosis and apoptosis involved. Autophagy also occurred in cells exposed to WAF mixtures at lower concentrations before any detectable cell death, indicating greater sensitivity to WAF exposure. The three types of cell behavior, namely necrosis, apoptosis and autophagy, may play different roles in oil spill-related respiratory disorders. PMID:22504303

  13. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    PubMed

    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.

  14. In vitro differentiation of a cloned bovine mammary epithelial cell.

    PubMed

    Rose, Michael T; Aso, Hisashi; Yonekura, Shinichi; Komatsu, Tokushi; Hagino, Akihiko; Ozutsumi, Kyouhei; Obara, Yoshiaki

    2002-08-01

    The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of alpha-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced alpha-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce alpha-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

  15. Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

    PubMed Central

    Centeno, A; Davis, C P; Cohen, M S; Warren, M M

    1983-01-01

    The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells. Images PMID:6132878

  16. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  17. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  18. Chronic Alcohol Exposure Renders Epithelial Cells Vulnerable to Bacterial Infection

    PubMed Central

    Wood, Stephen; Pithadia, Ravi; Rehman, Tooba; Zhang, Lijuan; Plichta, Jennifer; Radek, Katherine A.; Forsyth, Christopher; Keshavarzian, Ali; Shafikhani, Sasha H.

    2013-01-01

    Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier’s capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol’s deleterious effects on cells may be reversible. PMID:23358457

  19. Chronic alcohol exposure renders epithelial cells vulnerable to bacterial infection.

    PubMed

    Wood, Stephen; Pithadia, Ravi; Rehman, Tooba; Zhang, Lijuan; Plichta, Jennifer; Radek, Katherine A; Forsyth, Christopher; Keshavarzian, Ali; Shafikhani, Sasha H

    2013-01-01

    Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier's capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol's deleterious effects on cells may be reversible. PMID:23358457

  20. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  1. Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

    PubMed Central

    Thevenot, Paul T.; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D.; Chustz, Regina E.; Mahne, Sarah; Kelley, Matthew A.; Hebert, Valeria Y.; Dellinger, Barry; Dugas, Tammy R.; DeMayo, Francesco J.

    2013-01-01

    Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α–smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air–liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

  2. Expression and function of the protein tyrosine phosphatase receptor J (PTPRJ) in normal mammary epithelial cells and breast tumors.

    PubMed

    Smart, Chanel E; Askarian Amiri, Marjan E; Wronski, Ania; Dinger, Marcel E; Crawford, Joanna; Ovchinnikov, Dmitry A; Vargas, Ana Cristina; Reid, Lynne; Simpson, Peter T; Song, Sarah; Wiesner, Christiane; French, Juliet D; Dave, Richa K; da Silva, Leonard; Purdon, Amy; Andrew, Megan; Mattick, John S; Lakhani, Sunil R; Brown, Melissa A; Kellie, Stuart

    2012-01-01

    The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

  3. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  4. miR-124 regulates fetal pulmonary epithelial cell maturation

    PubMed Central

    Wang, Yang; Huang, Chaoqun; Chintagari, Narendranath Reddy; Xi, Dong; Weng, Tingting

    2015-01-01

    MicroRNAs are a family of small noncoding RNAs that regulate the expression of their target proteins at the posttranscriptional level. Their functions cover almost every aspect of cell physiology. However, the roles of microRNAs in fetal lung development are not completely understood. The objective of this study is to investigate the regulation and molecular mechanisms of alveolar epithelial cell maturation during fetal lung development by miR-124. We discovered that miR-124 was downregulated during rat fetal lung development and predominantly expressed in the epithelial cells at late stage of the lung development. Overexpression of miR-124 with an adenovirus vector led to the inhibition of epithelial maturation in rat fetal lung organ cultures and fetal alveolar epithelial type II cells, as demonstrated by a decrease in the type II cell marker expression and an increase in glycogen content. We further demonstrated by luciferase reporter assays that miR-124 inhibited the NF-κB, cAMP/PKA, and MAPK/ERK pathways. In addition, nuclear factor I/B (NFIB), a critical protein in fetal lung maturation, was validated as a direct target of miR-124. Furthermore, miR-124 expression was induced by the Wnt/β-catenin signaling pathway through a direct interaction of LEF1 and the miR-124 promoter region. We concluded that miR-124 downregulation is critical to fetal lung epithelial maturation and miR-124 inhibits this maturation process at least partially through the inhibition of NFIB. PMID:26071557

  5. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    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.

  6. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    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. PMID:27398446

  7. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    SciTech Connect

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang Zhang, Yi

    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.

  8. Notch Signaling in Meibomian Gland Epithelial Cell Differentiation

    PubMed Central

    Gidfar, Sanaz; Afsharkhamseh, Neda; Sanjari, Sara; Djalilian, Ali R.

    2016-01-01

    Purpose Notch1 was previously shown to play a critical role in murine meibomian gland function and maintenance. In this study, we have examined the expression and activation of Notch pathway in human meibomian gland epithelial cells in vitro. Methods An immortalized human meibomian gland epithelial cell (HMGEC) line was cultured under proliferative and differentiative conditions. Expression of Notch receptors and ligands were evaluated by quantitative PCR and Western blot. The effect of Notch inhibition and induction on oil production was also assessed. Results Human meibomian gland epithelial cell expressed Notch1, Notch2, Notch3, Jagged1, Jagged2, Delta-like 1, and Delta-like 3. The level of cleaved (activated) Notch1 strongly increased with differentiation. The expression of Notch3 was inversely correlated with proliferation. Induction and inhibition of Notch1 led to an increase and decrease in the amount of oil production, respectively. Conclusions Notch signaling appears to play an important role in human meibomian gland epithelial differentiation and oil production. This may provide a potential therapeutic pathway for treating meibomian gland dysfunction. PMID:26943148

  9. Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration

    PubMed Central

    Dudakov, Jarrod A.; Jenq, Robert R.; Velardi, Enrico; Young, Lauren F.; Smith, Odette M.; Lawrence, Gillian; Ivanov, Juliet A.; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L.; O'Rourke, Kevin P.; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas; Nieuwenhuis, Edward E.; Shroyer, Noah F.; Liu, Chen; Kolesnick, Richard

    2015-01-01

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration. PMID:26649819

  10. Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes.

    PubMed

    Svirshchevskaya, E V; Poltavtseva, R A; Beletskii, I P; Selezneva, I I; Sukhikh, G T

    2016-08-01

    We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin. PMID:27590756

  11. COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

    EPA Science Inventory

    Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

  12. Influence of ionizing radiation and 7,12-dimethylbenz(a)anthracene on the expression of mammary ductal dysplasia in mice

    SciTech Connect

    Ethier, S.P.

    1982-01-01

    These studies were undertaken to determine if altered growth potential of mammary epithelial cells could be detected in outgrowths derived from monodispersed mammary cells of virgin female BALB/c mice previously exposed to ionizing radiation or 7,12-dimethylbenz(a)anthracene (DMBA). Monodispersed mammary cells were obtained by enzymatic dissociation of mammary tissues of 12-week-old virgin female BALB/c mice. Twenty-four hours prior to cell dissociation, donor animals were exposed to either ..gamma..-ray irradiation or DMBA, while control donors were untreated. Mammary outgrowths were then derived from the donor cells by injecting either 10/sup 5/ or 10/sup 4/ cells into the gland free mammary fat pads of three-week-old virgin female BALB/c mice. In the initial studies all outgrowths were removed 10 weeks after injection of cells. The outgrowths that resulted were examined at the whole mount and histological level and were classified as having a normal ductal architecture or as having ductal dysplasia or alveolar adenosis. Outgrowths exhibiting ductal dysplasia were further classified as having mild or severe epithelial hyperplasia. The data indicated that treatment of donor animals with either ..gamma..-radiation or DMBA could result in an increased incidence of ductal dysplasias over control levels. Further, the incidence of lesions observed in all groups was influenced by the number of cells used to derive the outgrowths in that lesions were more frequent in outgrowths derived from 10/sup 4/ rather than 10/sup 5/ cells. The findings of these experiments indicate that acquisition of altered growth potential by mammary cells that results in expression of ductal dysplasia occurs soon after carcinogen treatment and that deriving mammary outgrowths from dissociated cells results in enhanced expression of these lesions.

  13. Midbody remnant licenses primary cilia formation in epithelial cells.

    PubMed

    Ott, Carolyn M

    2016-08-01

    Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model. PMID:27482049

  14. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    SciTech Connect

    Doupnik, C.A.; Leikauf, G.D. )

    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.

  15. Review: corneal epithelial stem cells, their niche and wound healing.

    PubMed

    Castro-Muñozledo, Federico

    2013-01-01

    Stem cells emerged as a concept during the second half of 19(th) century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet's membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  16. Review: Corneal epithelial stem cells, their niche and wound healing

    PubMed Central

    2013-01-01

    Stem cells emerged as a concept during the second half of 19th century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet’s membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  17. Curcuminoids and ω-3 fatty acids with anti-oxidants potentiate cytotoxicity of natural killer cells against pancreatic ductal adenocarcinoma cells and inhibit interferon γ production.

    PubMed

    Halder, Ramesh C; Almasi, Anasheh; Sagong, Bien; Leung, Jessica; Jewett, Anahid; Fiala, Milan

    2015-01-01

    Pancreatic cancer has a poor prognosis attributed in part to immune suppression and deactivation of natural killer (NK) cells. Curcuminoids have a potential for improving the therapy of pancreatic cancer given promising results in cancer models and a clinical trial, but their oral absorption is limited. Our objective in this study is to show curcuminoid anti-oncogenic effects alone and together with human NK cells. We tested curcuminoids in an emulsion of ω-3 fatty acids and anti-oxidants ("Smartfish") regarding their direct cytocidal effect and enhancement of the cytocidal activity of NK cells in pancreatic ductal adenocarcinoma (PDAC) cells (Mia Paca 2 and L3.6). Curcuminoids (at ≥10 μM) with ω-3 fatty acids and anti-oxidants or with the lipidic mediator resolvin D1 (RvD1) (26 nM) induced high caspase-3 activity in PDAC cells. Importantly, curcuminoids with ω-3 fatty acids and anti-oxidants or with RvD1 significantly potentiated NK cell cytocidal function and protected them against degradation. In a co-culture of cancer cells with NK cells, interferon-γ (IFN-γ) production by NK cells was not altered by ω-3 fatty acids with anti-oxidants or by RvD1 but was inhibited by curcuminoids. The inhibition was not eliminated by ω-3 fatty acids or RvD1 but was relieved by removing curcuminoids after adding NK cells. In conclusion, curcuminoids with ω-3 fatty acids and anti-oxidants or with RvD1 have increased cytotoxic activity on PDAC cells alone and with NK cells. The effects of curcuminoids with ω-3 fatty acids and anti-oxidants on pancreatic cancer will be investigated in a mouse model with humanized immune system.

  18. Curcuminoids and ω-3 fatty acids with anti-oxidants potentiate cytotoxicity of natural killer cells against pancreatic ductal adenocarcinoma cells and inhibit interferon γ production

    PubMed Central

    Halder, Ramesh C.; Almasi, Anasheh; Sagong, Bien; Leung, Jessica; Jewett, Anahid; Fiala, Milan

    2015-01-01

    Pancreatic cancer has a poor prognosis attributed in part to immune suppression and deactivation of natural killer (NK) cells. Curcuminoids have a potential for improving the therapy of pancreatic cancer given promising results in cancer models and a clinical trial, but their oral absorption is limited. Our objective in this study is to show curcuminoid anti-oncogenic effects alone and together with human NK cells. We tested curcuminoids in an emulsion of ω-3 fatty acids and anti-oxidants (“Smartfish”) regarding their direct cytocidal effect and enhancement of the cytocidal activity of NK cells in pancreatic ductal adenocarcinoma (PDAC) cells (Mia Paca 2 and L3.6). Curcuminoids (at ≥10 μM) with ω-3 fatty acids and anti-oxidants or with the lipidic mediator resolvin D1 (RvD1) (26 nM) induced high caspase-3 activity in PDAC cells. Importantly, curcuminoids with ω-3 fatty acids and anti-oxidants or with RvD1 significantly potentiated NK cell cytocidal function and protected them against degradation. In a co-culture of cancer cells with NK cells, interferon-γ (IFN-γ) production by NK cells was not altered by ω-3 fatty acids with anti-oxidants or by RvD1 but was inhibited by curcuminoids. The inhibition was not eliminated by ω-3 fatty acids or RvD1 but was relieved by removing curcuminoids after adding NK cells. In conclusion, curcuminoids with ω-3 fatty acids and anti-oxidants or with RvD1 have increased cytotoxic activity on PDAC cells alone and with NK cells. The effects of curcuminoids with ω-3 fatty acids and anti-oxidants on pancreatic cancer will be investigated in a mouse model with humanized immune system. PMID:26052286

  19. Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury.

    PubMed

    Gomperts, Brigitte N; Belperio, John A; Rao, P Nagesh; Randell, Scott H; Fishbein, Michael C; Burdick, Marie D; Strieter, Robert M

    2006-02-01

    Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases. PMID:16424223

  20. Vangl2 Regulates E-Cadherin in Epithelial Cells

    PubMed Central

    Nagaoka, Tadahiro; Inutsuka, Ayumu; Begum, Khadiza; hafiz, Khandakar musabbir bin; Kishi, Masashi

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

  1. Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells.

    PubMed

    Chanoux, Rebecca A; Shubin, Calla B; Robay, Amal; Suaud, Laurence; Rubenstein, Ronald C

    2013-10-01

    The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface. PMID:23885065

  2. Primary ductal adenocarcinoma of the lacrimal gland, associated with abundant intracytoplasmic lumens containing some eosinophilic hyaline globules: cytological, histological and ultrastructural findings.

    PubMed

    Min, Kyueng-Whan; Park, Hyung Kyu; Kim, Wook Youn; Kim, Wan-Seop; Lim, So Dug; Han, Hye Seung; Hwang, Tae Sook

    2014-10-01

    A primary ductal adenocarcinoma (PDA) of the lacrimal gland is a rare distinct subtype of an epithelial tumor arising in the lacrimal gland. PDA is the counterpart of salivary duct carcinoma (SDC) resembling an invasive ductal carcinoma (IDC) of the breast. In our case, PDA revealed histopathological and immunohistochemical results corresponding to SDC. Interestingly, the tumor cells showed intracytoplasmic vacuoles containing dense eosinophilic hyaline globules at light microscopy. Ultrastructurally, the tumor cells exhibited microvilli-lined intracytoplasmic lumen containing homogenous electron-dense secretory products. A previous study demonstrated that numerous intracytoplasmic lumens of tumor cells are favored breast malignant tumor, similar to the histopathology of PDA, rather than benign lesion. This characteristic finding may be meaningful to diagnose high grade epithelial tumors including PDA.

  3. Alveolar epithelial type II cell: defender of the alveolus revisited

    PubMed Central

    Fehrenbach, Heinz

    2001-01-01

    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. PMID:11686863

  4. Host epithelial geometry regulates breast cancer cell invasiveness

    PubMed Central

    Boghaert, Eline; Gleghorn, Jason P.; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C.; Nelson, Celeste M.

    2012-01-01

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment. PMID:23150585

  5. DNA analysis of epithelial cell suspensions

    SciTech Connect

    Wilson, J.S.; Johnson, N.F.; Holland, L.M.

    1985-01-01

    Cell suspensions of skin were obtained by animals exposed by skin painting of several crude oils. DNA analysis of these cell suspensions labeled with mithramycin provide determination of percentages of cells in the G/sub 1/, S and G/sub 2/M phases of the cell cycle. Data acquired showed differences from control animals occurring as early as 7 days after treatment and persisting through 21 days afterwards. There was histological evidence of erythema and hyperplasia in shale oil-exposed skins. Flow cytometric analysis of DNA content in shale-oil-exposed skin cells showed an increased percentage of cycling cells plus evidence of aneuploidy. Similar data from simply abraded skin showed increased percentages of cycling cells, but no aneuploidy. The shale-oil-exposed group, when compared to a standard petroleum-exposed group, had significantly increased percentages of cycling cells. This early indication of differing response to different complex mixtures was also seen in long-term skin exposures to these compounds. Similar analytical techniques were applied to tracheal cell suspensions from ozone-exposed rats. 12 refs., 4 figs., 4 tabs. (DT)

  6. To grow mouse mammary epithelial cells in culture

    PubMed Central

    1984-01-01

    Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures. PMID:6699079

  7. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  8. Helicobacter pylori increases proliferation of gastric epithelial cells.

    PubMed

    Fan, X G; Kelleher, D; Fan, X J; Xia, H X; Keeling, P W

    1996-01-01

    The direct and indirect effects of helicobacter pylori on cell kinetics of gastric epithelial cell line AGS were investigated by flow cytometric analysis of Ki-67 positive cells and by MTT assay. Flow cytometric analysis of Ki-67 positivity permits detection of cells that are in S-phase, whereas the MTT assay is a colometric measure of the number of viable cells. In the absence of added stimulants, 23.06 (4.88)% mean (SD) of AGS cells were Ki-67 positive. When cells were preincubated in the presence of H pylori, there was a significant increase in Ki-67 positivity (66.20 (7.89)%, p < 0.001). This increase was not seen in cells cultured in the presence of Campylobacter jejuni (24.63 (8.11)% or Escherichia coli (21.66 (9.78)%). Pre-incubation of AGS cells with supernatants from both H pylori and mitogen activated peripheral blood lymphocytes also increased the per cent of cells that were Ki-67 positive (72.93 (8.68) and 69.96 (12.35)%; p, 0.001) respectively. Similar results were also found in MTT assay. These data show that both H pylori directly and the immune/inflammatory response to H pylori indirectly can influence the rate of epithelial cell proliferation, suggesting this bacterium may be an initiating step in gastric carcinogenesis and an important co-carcinogenic factor in H pylori positive subjects.

  9. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  10. Clostridium difficile toxin A binding to human intestinal epithelial cells.

    PubMed

    Smith, J A; Cooke, D L; Hyde, S; Borriello, S P; Long, R G

    1997-11-01

    Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.

  11. Stiffness nanotomography of human epithelial cancer cells

    NASA Astrophysics Data System (ADS)

    Staunton, Jack R.; Doss, Bryant L.; Gilbert, C. Michael; Kasas, Sandor; Ros, Robert

    2012-02-01

    The mechanical stiffness of individual cells is important in both cancer initiation and metastasis. We present atomic force microscopy (AFM) based nanoindentation experiments on various human mammary and esophagus cell lines covering the spectrum from normal immortalized cells to highly metastatic ones. The combination of an AFM with a confocal fluorescence lifetime imaging microscope (FLIM) in conjunction with the ability to move the sample and objective independently allow for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. This enables us to correlate the mechanical properties with the point of indentation in the FLIM image. We are using force-volume measurements as well as force indentation curves on distinct points on the cells to compare the elastic moduli of the nuclei, nucleoli, and the cytoplasm, and how they vary within and between individual cells and cell lines. Further, a detailed analysis of the force-indentation curves allows study of the cells' mechanical properties at different indentation depths and to generate 3D elasticity maps.

  12. Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

    PubMed Central

    Lee, Wing-Kee; Dittmar, Thomas

    2014-01-01

    A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin. PMID:25407650

  13. Dpp signaling directs cell motility and invasiveness during epithelial morphogenesis.

    PubMed

    Ninov, Nikolay; Menezes-Cabral, Sofia; Prat-Rojo, Carla; Manjón, Cristina; Weiss, Alexander; Pyrowolakis, George; Affolter, Markus; Martín-Blanco, Enrique

    2010-03-23

    Tissue remodeling in development and disease involves the coordinated invasion of neighboring territories and/or the replacement of entire cell populations. Cell guidance, cell matching, transitions from passive to migratory epithelia, cell growth and death, and extracellular matrix remodeling all impinge on epithelial spreading. Significantly, the extracellular signals that direct these activities and the specific cellular elements and mechanisms regulated by these signals remain in most cases to be identified. To address these issues, we performed an analysis of histoblasts (Drosophila abdominal epithelial founder cells) on their transition from a dormant state to active migration replacing obsolete larval epidermal cells (LECs). We found that during expansion, Decapentaplegic (Dpp) secreted from surrounding LECs leads to graded pathway activation in cells at the periphery of histoblast nests. Across nests, Dpp activity confers differential cellular behavior and motility by modulating cell-cell contacts, the organization and activity of the cytoskeleton, and histoblast attachment to the substrate. Furthermore, Dpp also prevents the premature death of LECs, allowing the coordination of histoblast expansion to LEC delamination. Dpp signaling activity directing histoblast spreading and invasiveness mimics transforming growth factor-beta and bone morphogenetic proteins' role in enhancing the motility and invasiveness of cancer cells, resulting in the promotion of metastasis. PMID:20226662

  14. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

  15. Vimentin contributes to human mammary epithelial cell migration.

    PubMed

    Gilles, C; Polette, M; Zahm, J M; Tournier, J M; Volders, L; Foidart, J M; Birembaut, P

    1999-12-01

    Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.

  16. Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development.

    PubMed

    Assouline-Thomas, Béatrice; Ellis, Daniel; Petropavlovskaia, Maria; Makhlin, Julia; Ding, Jieping; Rosenberg, Lawrence

    2015-01-01

    Regeneration of β-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104-118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA. These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate.

  17. Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development.

    PubMed

    Assouline-Thomas, Béatrice; Ellis, Daniel; Petropavlovskaia, Maria; Makhlin, Julia; Ding, Jieping; Rosenberg, Lawrence

    2015-01-01

    Regeneration of β-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104-118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA. These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate. PMID:26558987

  18. Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells.

    PubMed

    Chen, Ying-Ting; Li, Wei; Hayashida, Yasutaka; He, Hua; Chen, Szu-Yu; Tseng, David Y; Kheirkhah, Ahmad; Tseng, Scheffer C G

    2007-08-01

    Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK(+)/Vim(+) or pan-CK(+)/Vim(-) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell-associated markers such as p63, Musashi-1, and ATP-binding cassette subfamily G2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus. Disclosure of potential conflicts of interest is found at the end of this article.

  19. GPER mediates estrogen-induced signaling and proliferations in human breast epithelial cells, and normal and malignant breast

    PubMed Central

    Scaling, Allison L.

    2014-01-01

    17β-estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized non-tumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant

  20. GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast.

    PubMed

    Scaling, Allison L; Prossnitz, Eric R; Hathaway, Helen J

    2014-06-01

    17β-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant

  1. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Vitamin C inhibit the proliferation, migration and epithelial-mesenchymal-transition of lens epithelial cells by destabilizing HIF-1α

    PubMed Central

    Zhao, Lin; Quan, Yanlong; Wang, Jianming; Wang, Feng; Zheng, Yuping; Zhou, Aiyi

    2015-01-01

    Posterior capsular opacification (PCO), the main complication of cataract surgery, is mainly caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the residual lens epithelial cells (LECs).Vitamin C was reported to reduce the risk of forming a cataract. However, there has been no study showing the association between vitamin C and PCO. In this study, we found that vitamin C could inhibit the migration and proliferation of human lens epithelial cells. We also found that vitamin C could increase the proline hydroxylation of HIF-1α and reduce the activity of HIF-1α. Moreover, vitamin C could not inhibit the activity of proline-mutant HIF-1α (402/564). Overexpression of wild-type HIF-1α or proline-mutant HIF-1α was found to increase the proliferation and migration of human lens epithelial cells. Differently, vitamin C could inhibit the proliferation and migration in wild-type HIF-1α-overexpressing lens epithelial cells but not the proline-mutant HIF-1α-overexpressing cells. Additionally, vitamin C was also found to inhibit the expression of EMT transcription factors TWIST. We then found that vitamin C could repress the EMT phenotypes induced by the overexpression of wild-type HIF-1α but not the proline-mutant HIF-1α. These results provide evidence that vitamin C plays a role in the repression of proliferation, migration, and EMT of human lens epithelial cells by destabilizing HIF-1α. PMID:26628999

  3. Proteomic profiling of rat lung epithelial cells induced by acrolein

    PubMed Central

    Sarkar, Poonam; Hayes, Barbara E.

    2009-01-01

    Aims Acrolein is a highly toxic unsaturated aldehyde and is also an endogenous byproduct produced from lipid peroxidation. It can be formed from the breakdown of certain pollutants in outdoor air or from burning tobacco or gasoline. Inhalation and dermal exposure to acrolein are extremely toxic to human tissue. Although it is known that acrolein is toxic to lung tissue, no studies have attempted to address the changes induced by acrolein on a global scale. Main methods In the present study we have attempted to address the changes in global protein expression induced by acrolein using proteomics analysis in rat lung epithelial cells. Key findings Our analysis reveals a comprehensive profiling of the proteins that includes a heterogeneous class of proteins and this compels one to consider that the toxic response to acrolein is very complex. There were 34 proteins that showed changes between the control cells and after acrolein treatment. The expression of 18 proteins was increased and the expression of 16 proteins was decreased following exposure to acrolein. We have further validated two differentially expressed proteins namely annexin II (ANXII) and prohibitin (PHB) in lung epithelial cells treated with acrolein. Significance Based on the results of the overall proteomic analysis, acrolein appears to induce changes in a diverse range of proteins suggesting a complex mechanism of acrolein-induced toxicity in lung epithelial cells. PMID:19490921

  4. Serratia marcescens is injurious to intestinal epithelial cells.

    PubMed

    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.

  5. In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

    PubMed

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

    Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue.

  6. MIA PaCa-2 and PANC-1 – pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors

    PubMed Central

    Gradiz, Rui; Silva, Henriqueta C.; Carvalho, Lina; Botelho, Maria Filomena; Mota-Pinto, Anabela

    2016-01-01

    Studies using cell lines should always characterize these cells to ensure that the results are not distorted by unexpected morphological or genetic changes possibly due to culture time or passage number. Thus, the aim of this study was to describe those MIA PaCa-2 and PANC-1 cell line phenotype and genotype characteristics that may play a crucial role in pancreatic cancer therapeutic assays, namely neuroendocrine chemotherapy and peptide receptor radionuclide therapy. Epithelial, mesenchymal, endocrine and stem cell marker characterization was performed by immunohistochemistry and flow cytometry, and genotyping by PCR, gene sequencing and capillary electrophoresis. MIA PaCa-2 (polymorphism) expresses CK5.6, AE1/AE3, E-cadherin, vimentin, chromogranin A, synaptophysin, SSTR2 and NTR1 but not CD56. PANC-1 (pleomorphism) expresses CK5.6, MNF-116, vimentin, chromogranin A, CD56 and SSTR2 but not E-cadherin, synaptophysin or NTR1. MIA PaCA-1 is CD24−, CD44+/++, CD326−/+ and CD133/1−, while PANC-1 is CD24−/+, CD44+, CD326−/+ and CD133/1−. Both cell lines have KRAS and TP53 mutations and homozygous deletions including the first 3 exons of CDKN2A/p16INK4A, but no SMAD4/DPC4 mutations or microsatellite instability. Both have neuroendocrine differentiation and SSTR2 receptors, precisely the features making them suitable for the therapies we propose to assay in future studies. PMID:26884312

  7. Left–right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    PubMed Central

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left–right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left–right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left–right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  8. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis.

    PubMed

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  9. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    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. PMID:26360608

  10. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  11. Oral epithelial cell responses to multispecies microbial biofilms.

    PubMed

    Peyyala, R; Kirakodu, S S; Novak, K F; Ebersole, J L

    2013-03-01

    This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms.

  12. Measles virus breaks through epithelial cell barriers to achieve transmission

    PubMed Central

    Takeda, Makoto

    2008-01-01

    Measles is a highly contagious disease that causes immunosuppression in patients. Measles virus infection has been thought to begin in the respiratory epithelium and then spread to lymphoid tissue. In this issue of the JCI, Leonard et al. provide data to suggest an alternative model of measles virus pathogenesis (see the related article beginning on page 2448). In human primary epithelial cells and rhesus monkeys in vivo, the authors show that initial infection of respiratory epithelium is not necessary for the virus to enter the host but that viral entry into epithelial cells via interaction of the virus with a receptor located on the basolateral side of the epithelium is required for viral shedding into the airway and subsequent transmission. PMID:18568081

  13. LOXL2 in epithelial cell plasticity and tumor progression.

    PubMed

    Cano, Amparo; Santamaría, Patricia G; Moreno-Bueno, Gema

    2012-09-01

    Several members of the lysyl oxidase family have recently emerged as important regulators of tumor progression. Among them, LOXL2 has been shown to be involved in tumor progression and metastasis of several tumor types, including breast carcinomas. Secreted LOXL2 participates in the remodeling of the extracellular matrix of the tumor microenvironment, in a similar fashion to prototypical lysyl oxidase. In addition, new intracellular functions of LOXL2 have been described, such as its involvement in the regulation of the epithelial-to-mesenchymal transition, epithelial cell polarity and differentiation mediated by transcriptional repression mechanisms. Importantly, intracellular (perinuclear) expression of LOXL2 is associated with poor prognosis and distant metastasis of specific tumor types, such as larynx squamous cell carcinoma and basal breast carcinomas. These recent findings open new avenues for the therapeutic utility of LOXL2.

  14. Epithelial Cell Polarity Determinant CRB3 in Cancer Development

    PubMed Central

    Li, Pingping; Mao, Xiaona; Ren, Yu; Liu, Peijun

    2015-01-01

    Cell polarity, which is defined as asymmetry in cell shape, organelle distribution and cell function, is essential in numerous biological processes, including cell growth, cell migration and invasion, molecular transport, and cell fate. Epithelial cell polarity is mainly regulated by three conserved polarity protein complexes, the Crumbs (CRB) complex, partitioning defective (PAR) complex and Scribble (SCRIB) complex. Research evidence has indicated that dysregulation of cell polarity proteins may play an important role in cancer development. Crumbs homolog 3 (CRB3), a member of the CRB complex, may act as a cancer suppressor in mouse kidney epithelium and mouse mammary epithelium. In this review, we focus on the current data available on the roles of CRB3 in cancer development. PMID:25552927

  15. Ivermectin inhibits growth of Chlamydia trachomatis in epithelial cells.

    PubMed

    Pettengill, Matthew A; Lam, Verissa W; Ollawa, Ikechukwu; Marques-da-Silva, Camila; Ojcius, David M

    2012-01-01

    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. PMID:23119027

  16. Characterization of foamy epithelial surface cells in the canine endometrium.

    PubMed

    Bartel, C; Tichy, A; Walter, I

    2014-06-01

    In mature bitches, endometrial epithelial surface cells modify function and corresponding morphology during the oestrous cycle. During late metoestrous, endometrial epithelial surface cells frequently accumulate fat and thereby adopt a foamy morphology. This cyclic appearance of foamy endometrial epithelial cells (fEECs) seems to be physiological in the dog, whereas in other species, it indicates pathological changes. Function of these fEECs has not been identified until now. Therefore, the aim of the study was to characterize the fEECs by means of transmission electron microscopy and immunohistochemistry. Different manifestations of fEECs were observed and analysed with regard to proliferative activity and presence of different epithelial adhesion molecules including PLEKHA7, β-catenin and E-cadherin. PLEKHA7 was restricted to the apical regions of the fEECs, whereas E-cadherin and β-catenin were demonstrated basolateral. The immunohistochemical detection of steroid hormone receptors demonstrated the responsiveness of the fEECs to steroid hormones. Intense progesterone receptor expression was observed in the fEECs indicating a high responsiveness to this hormone. Considering a potential function of the fEECs, we hypothesized that leptin, a hormone produced by other lipid-accumulating cells and described to be involved in reproduction, in particular during implantation, might also originate from the fEECs which was confirmed by immunohistochemical methods. Moreover, leptin receptor was found in fEECs indicating the fEECs as both, source and target for leptin. Therefore, we conclude that fEECs in the canine uterus have a potential role in early pregnancy events and that the different observed manifestations might simply reflect the variations of signs of pseudopregnancy among bitches.

  17. Role of medullary progenitor cells in epithelial cell migration and proliferation

    PubMed Central

    Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

    2014-01-01

    This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

  18. p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

    PubMed

    Hendley, Audrey M; Provost, Elayne; Bailey, Jennifer M; Wang, Yue J; Cleveland, Megan H; Blake, Danielle; Bittman, Ross W; Roeser, Jeffrey C; Maitra, Anirban; Reynolds, Albert B; Leach, Steven D

    2015-03-01

    The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development.

  19. Hyperoxia alters the mechanical properties of alveolar epithelial cells.

    PubMed

    Roan, Esra; Wilhelm, Kristina; Bada, Alex; Makena, Patrudu S; Gorantla, Vijay K; Sinclair, Scott E; Waters, Christopher M

    2012-06-15

    Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability. PMID:22467640

  20. TCDD alters medial epithelial cell differentiation during palatogenesis

    SciTech Connect

    Abbott, B.D.; Birnbaum, L.S. )

    1989-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of (3H)TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation.

  1. Expression of cell-cycle regulatory proteins BUBR1, MAD2, Aurora A, cyclin A and cyclin E in invasive ductal breast carcinomas.

    PubMed

    Du, Juan; Du, Qiang; Zhang, Yang; Sajdik, Constantin; Ruan, Yuan; Tian, Xin-xia; Fang, Wei-gang

    2011-06-01

    Cyclin A, cyclin E, BUBR1, MAD2 and Aurora A are all cell-cycle regulatory proteins and have been proven to play crucial roles in carcinogenesis. However, their expression patterns in invasive ductal breast carcinoma (IDBC) are controversial and unclear. In this study, we examined the expression status of these candidate proteins in a set of 117 invasive ductal carcinomas, and evaluated their associations with known clinicopathological parameters and the expressions of estrogen receptor, progesterone receptor, Ki-67 and Her-2. Univariate and multivariate data analyses both displayed that positive BUBR1 expression was associated with a high Ki-67 labeling index, and negative MAD2 expression was associated with Her-2 overexpression. Positive BUBR1 expression was also associated with a high histological tumor grade in univariate analysis, but not in multivariate analysis. In addition, high Aurora A expression was weakly associated with lymph node metastasis, and cyclin A was strongly associated with the expression of cyclin E in both univariate and multivariate models. In conclusion, this study suggests that evaluation of BUBR1, MAD2 and Aurora A expression levels is likely to improve accuracy of prognostic predictions in IDBC.

  2. Elastic properties of epithelial cells probed by atomic force microscopy.

    PubMed

    Brückner, Bastian R; Janshoff, Andreas

    2015-11-01

    Cellular mechanics plays a crucial role in many biological processes such as cell migration, cell growth, embryogenesis, and oncogenesis. Epithelia respond to environmental cues comprising biochemical and physical stimuli through defined changes in cell elasticity. For instance, cells can differentiate between certain properties such as viscoelasticity or topography of substrates by adapting their own elasticity and shape. A living cell is a complex viscoelastic body that not only exhibits a shell architecture composed of a membrane attached to a cytoskeleton cortex but also generates contractile forces through its actomyosin network. Here we review cellular mechanics of single cells in the context of epithelial cell layers responding to chemical and physical stimuli. This article is part of a Special Issue entitled: Mechanobiology. PMID:26193077

  3. Characterization of goldfish fin cells in culture: some evidence of an epithelial cell profile.

    PubMed

    Mauger, P-E; Labbé, C; Bobe, J; Cauty, C; Leguen, I; Baffet, G; Le Bail, P-Y

    2009-03-01

    Comprehensive characterization of cultured cells in fish was little explored and cell origin is often deduced from morphological analogies with either epithelial of fibroblastic cells. This study aims to characterize cell origin in goldfish fin culture using morphological, immunochemical, and molecular approaches. Time lapse analysis revealed that cultured cell morphology changed within minutes. Therefore, cell morphology cannot predict whether cells are from fibroblastic or epithelial origin. The labeling pattern of heterologous anti-cytokeratin and anti-vimentin antibodies against goldfish epithelial cells and fibroblasts was first tested on skin sections and the corresponding labeling of the cultured cells was analyzed. No cell origin specificity could be obtained with the chosen antibodies. In the molecular approach, detection levels of three cytokeratin (CauK8-IIS, CauK49-IE and CauK50-Ie) and one vimentin transcripts were assessed on skin and fin samples. Specificity for epithelial cells of the most abundant mRNA, CauK49-Ie, was thereafter validated on skin sections by in situ hybridization. The selected markers were used afterwards to characterize fin cultures. CauK49-IE riboprobe labeled every cell in young cultures whereas no labeling was observed in older cultures. Accordingly, CauK49-IE transcript levels decreased after 15 days culture while CauK8-IIS ones increased. The use of homologous marker gave evidence that young cultured cells from goldfish fin are homogeneously of epithelial type and that cell characteristics may change over culture time.

  4. Comparison of functional limbal epithelial stem cell isolation methods.

    PubMed

    López-Paniagua, Marina; Nieto-Miguel, Teresa; de la Mata, Ana; Dziasko, Marc; Galindo, Sara; Rey, Esther; Herreras, José M; Corrales, Rosa M; Daniels, Julie T; Calonge, Margarita

    2016-05-01

    The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either

  5. 293 cells express both epithelial as well as mesenchymal cell adhesion molecules

    PubMed Central

    INADA, MASAKAZU; IZAWA, GENYA; KOBAYASHI, WAKAKO; OZAWA, MASAYUKI

    2016-01-01

    The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells. PMID:27121032

  6. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles

    PubMed Central

    Chiabotto, Giulia; Bruno, Stefania; Collino, Federica

    2016-01-01

    Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs. PMID:27409796

  7. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells.

    PubMed

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  8. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells

    PubMed Central

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  9. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.

    PubMed

    Luissint, Anny-Claude; Parkos, Charles A; Nusrat, Asma

    2016-10-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte-epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  10. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  11. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  12. Studies in human skin epithelial cell carcinogenesis

    SciTech Connect

    Lehman, T.A.

    1987-01-01

    Metabolism and DNA adduct formation of benzo(a)pyrene (BP) by human epidermal keratinocytes pretreated with inhibitors or inducer of cytochrame P450 was studied. To study DNA adduct analysis, cultures were pretreated as described above, and then treated with non-radiolabeled BP. DNA was prepared from these cultures, digested to the nucleotide level, and /sup 32/P-postlabeled for adduct analysis. Cultures pretreated with BHA, 7,8-BF or disulfiralm formed significantly fewer BPDE I-dB adducts than non-pretreated cultures, while cultures pretreated with MeBHA formed more BPDE-I-dG adducts. MeBHA increased BP activation and adduct formation inhuman keratinocyte in cultures by inducing a specific isoenzyme of cytochrome P450 which preferentially increases the oxidative metabolism of BP to 7,8 diol BP and 7,8 diol BP to BPDE I. To approximate an in vivo human system, metabolism of BPDE I by human skin xenografts treated with cell cycles modulators was studied. When treated with BPDE I, specific carcinogen-DNA adducts were formed. Separation and identification of these adducts by the /sup 32/P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts.

  13. Hertwig's epithelial root sheath cells do not transform into cementoblasts in rat molar cementogenesis.

    PubMed

    Yamamoto, Tsuneyuki; Takahashi, Shigeru

    2009-12-01

    It is generally accepted that cementoblasts originate in the process of differentiation of the mesenchymal cells of the dental follicle. Recently, a different hypothesis for the origin of cementoblasts has been proposed. Hertwig's epithelial root sheath cells undergo the epithelial-mesenchymal transformation to differentiate into cementoblasts. To elucidate whether the epithelial-mesenchymal transformation occurs in the epithelial sheath, developing rat molars were examined by keratin-vimentin and Runx2 (runt-related transcription factor 2)-keratin double immunostaining. In both acellular and cellular cementogenesis, epithelial sheath and epithelial cells derived from the epithelial sheath expressed keratin, but did not express vimentin or Runx2. Dental follicle cells and cementoblasts, however, expressed vimentin and Runx2, but did not express keratin. No cells showed coexisting keratin-vimentin or Runx2-keratin staining. These findings suggest that there is no intermediate phenotype transforming epithelial to mesenchymal cells, and that epithelial sheath cells do not generate mineralized tissue. This study concludes that the epithelial-mesenchymal transformation does not occur in Hertwig's epithelial root sheath in rat acellular or cellular cementogenesis and that the dental follicle is the origin of cementoblasts, as has been proposed in the original hypothesis. PMID:19716687

  14. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  15. Thymic epithelial cells: working class heroes for T cell development and repertoire selection.

    PubMed

    Anderson, Graham; Takahama, Yousuke

    2012-06-01

    The thymus represents an epithelial-mesenchymal tissue, anatomically structured into discrete cortical and medullary regions that contain phenotypically and functionally distinct stromal cells, as well as thymocytes at defined stages of maturation. The stepwise progression of thymocyte development seems to require serial migration through these distinct thymic regions, where interactions with cortical thymic epithelial cell (cTEC) and medullary thymic epithelial cell (mTEC) subsets take place. Recent work on TEC subsets provides insight into T cell development and selection, such as the importance of tumour necrosis factor (TNF) receptor superfamily members in thymus medulla development, and the specialised antigen processing/presentation capacity of the thymic cortex for positive selection. Here, we summarise current knowledge on the development and function of the thymic microenvironment, paying particular attention to the cortical and medullary epithelial compartments.

  16. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

  17. Incremental responses to light recorded from pigment epithelial cells and horizontal cells of the cat retina

    PubMed Central

    Steinberg, Roy H.

    1971-01-01

    1. Rod-dependent incremental responses were recorded intracellularly in both pigment epithelial cells and horizontal cells of the cat retina. They were elicited by test flashes which were superimposed on background flashes after a delay. 2. In pigment epithelial cells smaller test responses were produced as background intensity was raised. The incremental sensitivity function was linear for about 1·4 log units, with a slope of 0·86, and the approach of saturation occurred at about 2·5 log td scotopic. 3. The amplitude of pigment epithelial test responses could be estimated from the dark-adapted amplitude—log intensity function obtained with single flashes. Test flashes produced the voltage increment predicted by the slope of this function just above the point on the curve equal to the background intensity. The pigment epithelial response to a test flash, therefore, is the response expected if the background were presented alone and made more intense by the amount of the test flash. 4. Rod-dependent incremental sensitivity functions of horizontal cells closely resembled the ones obtained from pigment epithelial cells. 5. It was concluded that the adaptive effects observed in pigment epithelial cells originated in individual rods. These effects arose from the compressive nature of the dark-adapted amplitude—intensity function. In horizontal cell responses these effects may be modified by the failure of the background response to maintain its initial voltage. PMID:5571955

  18. Oral microbial biofilm stimulation of epithelial cell responses.

    PubMed

    Peyyala, Rebecca; Kirakodu, Sreenatha S; Novak, Karen F; Ebersole, Jeffrey L

    2012-04-01

    Oral bacterial biofilms trigger chronic inflammatory responses in the host that can result in the tissue destructive events of periodontitis. However, the characteristics of the capacity of specific host cell types to respond to these biofilms remain ill-defined. This report describes the use of a novel model of bacterial biofilms to stimulate oral epithelial cells and profile select cytokines and chemokines that contribute to the local inflammatory environment in the periodontium. Monoinfection biofilms were developed with Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis on rigid gas-permeable contact lenses. Biofilms, as well as planktonic cultures of these same bacterial species, were incubated under anaerobic conditions with a human oral epithelial cell line, OKF4, for up to 24h. Gro-1α, IL1α, IL-6, IL-8, TGFα, Fractalkine, MIP-1α, and IP-10 were shown to be produced in response to a range of the planktonic or biofilm forms of these species. P. gingivalis biofilms significantly inhibited the production of all of these cytokines and chemokines, except MIP-1α. Generally, the biofilms of all species inhibited Gro-1α, TGFα, and Fractalkine production, while F. nucleatum biofilms stimulated significant increases in IL-1α, IL-6, IL-8, and IP-10. A. naeslundii biofilms induced elevated levels of IL-6, IL-8 and IP-10. The oral streptococcal species in biofilms or planktonic forms were poor stimulants for any of these mediators from the epithelial cells. The results of these studies demonstrate that oral bacteria in biofilms elicit a substantially different profile of responses compared to planktonic bacteria of the same species. Moreover, certain oral species are highly stimulatory when in biofilms and interact with host cell receptors to trigger pathways of responses that appear quite divergent from individual bacteria.

  19. Human epithelial cell cultures from superficial limbal explants

    PubMed Central

    Basli, E.; Goldschmidt, P.; Pecha, F.; Chaumeil, C.; Laroche, L.; Borderie, V.

    2011-01-01

    Purpose To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer. Methods Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR). Results The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm2 and 429 µm2, and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human

  20. Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

    PubMed

    Martínez-Martínez, Eduardo; Uribe-Querol, Eileen; Galván-Hernández, Claudio I; Gutiérrez-Ospina, Gabriel

    2016-01-01

    We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology. PMID:27431250

  1. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  2. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  3. Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

    PubMed

    Hatakeyama, S; Gao, Y H; Ohara-Nemoto, Y; Kataoka, H; Satoh, M

    1997-07-01

    Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either. PMID:9247707

  4. Polycomb repressor complex 1 promotes gene silencing through H2AK119 mono-ubiquitination in acinar-to-ductal metaplasia and pancreatic cancer cells

    PubMed Central

    Reinhard, Tobias; Popp, Anna; Schäffer, Isabell; Raulefs, Susanne; Kong, Bo; Esposito, Irene

    2016-01-01

    Acinar-to-ductal metaplasia (ADM) occurring in cerulein-mediated pancreatitis or in oncogenic Kras-driven pancreatic cancer development is accompanied by extensive changes in the transcriptional program. In this process, acinar cells shut down the expression of acinar specific differentiation genes and re-express genes usually found in embryonic pancreatic progenitor cells. Previous studies have demonstrated that a loss of acinar-specific transcription factors sensitizes the cells towards oncogenic transformation, ultimately resulting in cancer development. However, the mechanism behind the transcriptional silencing of acinar cell fate genes in ADM and pancreatic cancer is largely unknown. Here, we analyzed whether elevated levels of the polycomb repressor complex 1 (PRC1) components Bmi1 and Ring1b and their catalyzed histone modification H2AK119ub in ADMs and tumor cells, are responsible for the mediation of acinar gene silencing. Therefore, we performed chromatin-immunoprecipitation in in vitro generated ADMs and isolated murine tumor cells against the repressive histone modifications H3K27me3 and H2AK119ub. We established that the acinar transcription factor complex Ptf1-L is epigenetically silenced in ADMs as well as in pancreatic tumor cells. For the first time, this work presents a possible mechanism of acinar gene silencing, which is an important prerequisite in the initiation and maintenance of a dedifferentiated cell state in ADMs and tumor cells. PMID:26716510

  5. Interleukin-6 induces epithelial-mesenchymal transition in human intrahepatic biliary epithelial cells

    PubMed Central

    JIANG, GUI-XING; CAO, LI-PING; KANG, PENG-CHENG; ZHONG, XIANG-YU; LIN, TIAN-YU; CUI, YUN-FU

    2016-01-01

    The aim of the present study was to determine the role of interleukin-6 (IL-6) in the epithelial-mesenchymal transition (EMT) of human intrahepatic biliary epithelial cell (HIBEC) lines in vitro. HIBECs were stimulated with IL-6 at concentrations of 0, 10, 20, 50 and 100 µg/l for 24 h. A wound healing and Transwell assay were performed to determine the migratory and invasive capacity of HIBECs, respectively. Following 24 h of incubation, IL-6 at 10 and 20 µg/l significantly increased the number of migrated and invaded cells (P<0.05), while stimulation with 50 and 100 µg/l IL-6 resulted in a further increase of the migratory and invasive capacity compared to that in all other groups (P<0.05). Furthermore, reverse-transcription quantitative polymerase chain reaction and western blot analyses were used to detect the mRNA and protein expression of EMT markers E-cadherin and vimentin in HIBECs. Decreased mRNA levels of E-cadherin accompanied by higher mRNA levels of vimentin were observed in the 10, 20, 50, 100 µg/l IL-6 groups compared to those in the 0 µg/l group (all P<0.05). Furthermore, the protein expression of E-cadherin was decreased, while that of vimentin was increased in the 50 and 100 µg/l IL-6 groups compared to those in the 0, 10 and 20 µg/l IL-6 groups (all P<0.05). The present study therefore indicated that IL-6 promoted the process of EMT in HIBECs as characterized by increased migration and invasion of HIBECs and the typical changes in mRNA and protein expression of the EMT markers E-cadherin and vimentin. PMID:26708270

  6. Isolating Epithelial and Epithelial-to-Mesenchymal Transition Populations from Primary Tumors by Fluorescence-Activated Cell Sorting.

    PubMed

    Aiello, Nicole M; Rhim, Andrew D; Stanger, Ben Z

    2016-01-01

    Transgenic mice that express conditional reporters allow for the isolation of specific cell lineages. These cells can be further stratified by gene expression and collected by fluorescence-activated cell sorting (FACS) for further analysis. Using Cre-recombinase (Cre) technology we have generated a transgenic mouse line termed PKCY in which all pancreatic epithelial cells and therefore all pancreatic cancer cells are constitutively labeled with yellow fluorescent protein (YFP). We have used immunofluorescent staining for E-cadherin to divide the YFP(+) tumor population into epithelial cells (E-cadherin positive) and cells that have undergone an epithelial-to-mesenchymal transition (EMT; E-cadherin negative). This protocol describes how to prepare a tumor sample for FACS, with an emphasis on separating epithelial and EMT populations. These cells can then be used for a number of applications including, but not limited to, the generation of cell lines, gene-expression analysis by quantitative polymerase chain reaction (qPCR) or RNA sequencing, DNA sequencing, chromatin immunoprecipitation, and western blots. PMID:26729901

  7. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland.

  8. Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

    NASA Astrophysics Data System (ADS)

    Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

    2006-07-01

    A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

  9. Sensing, signaling and sorting events in kidney epithelial cell physiology.

    PubMed

    Brown, Dennis; Breton, Sylvie; Ausiello, Dennis A; Marshansky, Vladimir

    2009-03-01

    The kidney regulates body fluid, ion and acid/base homeostasis through the interaction of a host of channels, transporters and pumps within specific tubule segments, specific cell types and specific plasma membrane domains. Furthermore, renal epithelial cells have adapted to function in an often unique and challenging environment that includes high medullary osmolality, acidic pHs, variable blood flow and constantly changing apical and basolateral 'bathing' solutions. In this review, we focus on selected protein trafficking events by which kidney epithelial cells regulate body fluid, ion and acid-base homeostasis in response to changes in physiological conditions. We discuss aquaporin 2 and G-protein-coupled receptors in fluid and ion balance, the vacuolar H(+)-adenosine triphosphatase (V-ATPase) and intercalated cells in acid/base regulation and acidification events in the proximal tubule degradation pathway. Finally, in view of its direct role in vesicle trafficking that we outline in this study, we propose that the V-ATPase itself should, under some circumstances, be considered a fourth category of vesicle 'coat' protein (COP), alongside clathrin, caveolin and COPs. PMID:19170982

  10. Sensing, signaling and sorting events in kidney epithelial cell physiology

    PubMed Central

    Brown, Dennis; Breton, Sylvie; Ausiello, Dennis A.; Marshansky, Vladimir

    2010-01-01

    The kidney regulates body fluid, ion, and acid/base homeostasis via the interaction of a host of channels, transporters, and pumps within specific tubule segments, specific cell types, and specific plasma membrane domains. Furthermore, renal epithelial cells have adapted to function in an often unique and challenging environment that includes high medullary osmolality, acidic pHs, variable blood flow and constantly changing apical and basolateral “bathing” solutions. In this review, we focus on selected protein trafficking events by which kidney epithelial cells regulate body fluid, ion and acid-base homeostasis in response to changes in physiological conditions. We discuss aquaporin 2 and G-protein coupled receptors (GPCRs) in fluid and ion balance, the V-ATPase and intercalated cells (IC) in acid/base regulation, and acidification events in the proximal tubule degradation pathway. Finally, in view of its direct role in vesicle trafficking that we outline here, we propose that the V-ATPase itself should, under some circumstances, be considered as a fourth category of vesicle “coat” protein, alongside clathrin, caveolin, and COPs. PMID:19170982

  11. Organization of the cytokeratin network in an epithelial cell.

    PubMed

    Portet, Stéphanie; Arino, Ovide; Vassy, Jany; Schoëvaërt, Damien

    2003-08-01

    The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters.

  12. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival.

    PubMed

    Lamberti, Yanina; Gorgojo, Juan; Massillo, Cintia; Rodriguez, Maria E

    2013-12-01

    Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

  13. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    PubMed

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  14. Relation of smoking to immunoreactive endothelin in the bronchiolar epithelial cells.

    PubMed Central

    Shokeir, M. O.; Paré, P.; Wright, J. L.

    1994-01-01

    BACKGROUND--Endothelin is a potent bronchoconstrictor which appears to be important in asthma. To ascertain whether cigarette smoking is associated with any alteration in the proportion of bronchiolar epithelial cells which express endothelin immunoreactivity, the airways in the lungs of non-smokers and smokers were analysed. Since an increase in immunoreactivity has been found in the bronchial epithelial cells of asthmatic subjects, cigarette smokers with and without evidence of airway hyperresponsiveness were also selected. METHODS--A point counting method which examined the proportion of endothelin immunoreactive epithelial cells in membranous and respiratory bronchioles was used. RESULTS--Neither smoking itself nor evidence of airway hyperresponsiveness altered the percentage of endothelin immunoreactive epithelial cells in the membraneous and respiratory bronchioles. CONCLUSIONS--Cigarette smoke does not induce endothelin production in bronchiolar epithelial cells, and the airway hyperresponsiveness seen in some patients with lung disease induced by cigarette smoking is not related to exaggerated endothelin production in epithelial cells. PMID:8091324

  15. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  16. Plasticity of epithelial stem cells in tissue regeneration

    PubMed Central

    Blanpain, Cédric; Fuchs, Elaine

    2015-01-01

    Tissues rely upon stem cells for homeostasis and repair. Recent studies show that the fate and multilineage potential of epithelial stem cells can change depending on whether a stem cell exists within its resident niche and responds to normal tissue homeostasis, whether it is mobilized to repair a wound, or whether it is taken from its niche and challenged to de novo tissue morphogenesis after transplantation. In this Review, we discuss how different populations of naturally lineage-restricted stem cells and committed progenitors can display remarkable plasticity and reversibility and reacquire long-term self-renewing capacities and multilineage differentiation potential during physiological and regenerative conditions. We also discuss the implications of cellular plasticity for regenerative medicine and for cancer. PMID:24926024

  17. Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

    PubMed Central

    1994-01-01

    Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed. PMID:7798314

  18. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

    SciTech Connect

    Roberts, Joan E. Wielgus, Albert R. Boyes, William K. Andley, Usha Chignell, Colin F.

    2008-04-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C{sub 60}(OH){sub 22-26}] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C{sub 60}(OH){sub 22-26} in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 {mu}M. Exposure to either UVA or visible light in the presence of > 5 {mu}M fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 {mu}M lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein {alpha}-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C{sub 60}(OH){sub 22-26} is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo.

  19. In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

    PubMed Central

    Hu, Han; Wang, Jiaqi; Bu, Dengpan; Wei, Hongyang; Zhou, Linyun; Li, Fadi; Loor, Juan J.

    2009-01-01

    Background The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. Methodology Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. Principal Findings The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. Conclusions The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs). PMID:19888476

  20. Protein kinase C expression in salivary gland acinar epithelial cells in non-obese diabetic mice, an experimental model for Sjögren's syndrome.

    PubMed

    Tensing, E-K; Ma, J; Hukkanen, M; Fox, H S; Li, T-F; Törnwall, J; Konttinen, Y T

    2005-01-01

    We planned to investigate the expression of protein kinase C (PKC) isoforms in acinar epithelial cells of salivary glands in the non-obese diabetic (NOD) mouse to find out if they develop changes of the PKC system like those seen in the human counterpart, i.e. in Sjögren's syndrome. Parotid, submandibular, and sublingual glands from NOD and control BALB/c mice were stained with a panel of monoclonal antibodies directed against conventional (alpha, beta, and gamma), novel (delta, epsilon, and theta), and atypical (lambda and iota) PKC isoforms using the streptavidin/HRP method. Similarly to human labial salivary glands, acinar epithelial cells of the healthy control BALB/c mice contained two of the conventional PKC isoforms, alpha and beta. Acinar and ductal epithelial cells also contained the atypical PKC isoforms lambda and iota. PKC isoforms gamma, delta, epsilon, and theta were not found. NOD mice which displayed focal sialadenitis contained the same conventional and atypical PKC isoforms. The acinar cells in NOD mice, in contrast to the Sjögren's syndrome patients, did not lack PKC alpha or beta. On the contrary, PKC alpha and beta staining was stronger than in the control BALB/c mice. The present results demonstrate that both conventional and atypical PKC isoforms participate in the salivary epithelial cell biology and that there are mouse strain-associated and/or disease state-associated changes in their expression. The lack of PKC alpha and beta isoforms found in Sjögren's syndrome was not reproduced in NOD mice, which discloses one more difference between the human disease and its NOD mouse model.

  1. Cell crawling mediates collective cell migration to close undamaged epithelial gaps

    PubMed Central

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C.; Sheetz, Michael P.; Trepat, Xavier; Ladoux, Benoît

    2012-01-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  2. Cell crawling mediates collective cell migration to close undamaged epithelial gaps.

    PubMed

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C; Sheetz, Michael P; Trepat, Xavier; Ladoux, Benoît

    2012-07-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  3. Ductal adenoma of the breast: a lesion which can mimic carcinoma.

    PubMed

    Azzopardi, J G; Salm, R

    1984-09-01

    Twenty-four cases of a solid benign tumour of breast ducts are described, for which we propose the name 'ductal adenoma'. The lesion consists of a single nodule or multiple nodules involving medium size and small ducts, but not major subareolar ducts. It presents as a palpable lump, and is not associated with a nipple discharge. Clinically, radiologically and macroscopically, it can simulate malignancy because of its occurrence in older age groups, frequent microcalcification and the firmness and irregularity of many lesions. Fibrous sclerosis sometimes results in distortion with apparent invasion of surrounding tissue. It can be mistaken for carcinoma both on frozen and paraffin sections. Differentiation into epithelial and myoepithelial cells is the most reliable criterion in the recognition of this lesion as benign. It has microscopic affinities with ductal papilloma, on the one hand, and with salivary-type adenoma, on the other. Ductal adenoma constitutes the third major type of adenoma in the breast, in addition to the already widely recognized nipple adenoma and tubular adenoma.

  4. Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells

    PubMed Central

    Pantenburg, Birte; Castellanos-Gonzalez, Alejandro; Dann, Sara M.; Connelly, Rhykka L.; Lewis, Dorothy E.; Ward, Honorine D.; Clinton White, A.

    2010-01-01

    Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4+ T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8+ T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8+ T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8+ T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite. PMID:20348507

  5. GPRC5A is a potential oncogene in pancreatic ductal adenocarcinoma cells that is upregulated by gemcitabine with help from HuR.

    PubMed

    Zhou, H; Telonis, A G; Jing, Y; Xia, N L; Biederman, L; Jimbo, M; Blanco, F; Londin, E; Brody, J R; Rigoutsos, I

    2016-01-01

    GPRC5A is an orphan G-protein coupled receptor with an intriguing dual behavior, acting as an oncogene in some cancers and as a tumor suppressor in other cancers. In the pancreatic cancer context, very little is known about GPRC5A. By analyzing messenger RNA (mRNA) expression data from 675 human cancer cell lines and 10 609 samples from The Cancer Genome Atlas (TCGA) we found that GPRC5A's abundance in pancreatic cancer is highest (cell lines) or second highest (TCGA) among all tissues and cancer types. Further analyses of an independent set of 252 pancreatic normal and cancer samples showed GPRC5A mRNA to be more than twofold upregulated in primary tumor samples compared with normal pancreas (P-value<10(-5)), and even further upregulated in pancreatic cancer metastases to various organs (P-value=0.0021). Immunostaining of 208 cores (103 samples) of a tissue microarray showed generally low expression of GPRC5A protein in normal pancreatic ductal cells; on the other hand, in primary and metastatic samples, GPRC5A protein levels were dramatically increased in pancreatic ductal cells. In vitro studies of multiple pancreatic cancer cell lines showed that an increase in GPRC5A protein levels promoted pancreatic cancer cell growth and migration. Unexpectedly, when we treated pancreatic cancer cell lines with gemcitabine (2',2'-difluorodeoxycytidine), we observed an increase in GPRC5A protein abundance. On the other hand, when we knocked down GPRC5A we sensitized pancreatic cancer cells to gemcitabine. Through further experimentation we showed that the monotonic increase in GPRC5A protein levels that we observe for the first 18 h following gemcitabine treatment results from interactions between GPRC5A's mRNA and the RNA-binding protein HuR, which is an established key mediator of gemcitabine's efficacy in cancer cells. As we discovered, the interaction between GPRC5A and HuR is mediated by at least one HuR-binding site in GPRC5A's mRNA. Our findings indicate that GPRC

  6. GPRC5A is a potential oncogene in pancreatic ductal adenocarcinoma cells that is upregulated by gemcitabine with help from HuR.

    PubMed

    Zhou, H; Telonis, A G; Jing, Y; Xia, N L; Biederman, L; Jimbo, M; Blanco, F; Londin, E; Brody, J R; Rigoutsos, I

    2016-07-14

    GPRC5A is an orphan G-protein coupled receptor with an intriguing dual behavior, acting as an oncogene in some cancers and as a tumor suppressor in other cancers. In the pancreatic cancer context, very little is known about GPRC5A. By analyzing messenger RNA (mRNA) expression data from 675 human cancer cell lines and 10 609 samples from The Cancer Genome Atlas (TCGA) we found that GPRC5A's abundance in pancreatic cancer is highest (cell lines) or second highest (TCGA) among all tissues and cancer types. Further analyses of an independent set of 252 pancreatic normal and cancer samples showed GPRC5A mRNA to be more than twofold upregulated in primary tumor samples compared with normal pancreas (P-value<10(-5)), and even further upregulated in pancreatic cancer metastases to various organs (P-value=0.0021). Immunostaining of 208 cores (103 samples) of a tissue microarray showed generally low expression of GPRC5A protein in normal pancreatic ductal cells; on the other hand, in primary and metastatic samples, GPRC5A protein levels were dramatically increased in pancreatic ductal cells. In vitro studies of multiple pancreatic cancer cell lines showed that an increase in GPRC5A protein levels promoted pancreatic cancer cell growth and migration. Unexpectedly, when we treated pancreatic cancer cell lines with gemcitabine (2',2'-difluorodeoxycytidine), we observed an increase in GPRC5A protein abundance. On the other hand, when we knocked down GPRC5A we sensitized pancreatic cancer cells to gemcitabine. Through further experimentation we showed that the monotonic increase in GPRC5A protein levels that we observe for the first 18 h following gemcitabine treatment results from interactions between GPRC5A's mRNA and the RNA-binding protein HuR, which is an established key mediator of gemcitabine's efficacy in cancer cells. As we discovered, the interaction between GPRC5A and HuR is mediated by at least one HuR-binding site in GPRC5A's mRNA. Our findings indicate that GPRC

  7. GPRC5A is a potential oncogene in pancreatic ductal adenocarcinoma cells that is upregulated by gemcitabine with help from HuR

    PubMed Central

    Zhou, H; Telonis, A G; Jing, Y; Xia, N L; Biederman, L; Jimbo, M; Blanco, F; Londin, E; Brody, J R; Rigoutsos, I

    2016-01-01

    GPRC5A is an orphan G-protein coupled receptor with an intriguing dual behavior, acting as an oncogene in some cancers and as a tumor suppressor in other cancers. In the pancreatic cancer context, very little is known about GPRC5A. By analyzing messenger RNA (mRNA) expression data from 675 human cancer cell lines and 10 609 samples from The Cancer Genome Atlas (TCGA) we found that GPRC5A's abundance in pancreatic cancer is highest (cell lines) or second highest (TCGA) among all tissues and cancer types. Further analyses of an independent set of 252 pancreatic normal and cancer samples showed GPRC5A mRNA to be more than twofold upregulated in primary tumor samples compared with normal pancreas (P-value<10−5), and even further upregulated in pancreatic cancer metastases to various organs (P-value=0.0021). Immunostaining of 208 cores (103 samples) of a tissue microarray showed generally low expression of GPRC5A protein in normal pancreatic ductal cells; on the other hand, in primary and metastatic samples, GPRC5A protein levels were dramatically increased in pancreatic ductal cells. In vitro studies of multiple pancreatic cancer cell lines showed that an increase in GPRC5A protein levels promoted pancreatic cancer cell growth and migration. Unexpectedly, when we treated pancreatic cancer cell lines with gemcitabine (2′,2′-difluorodeoxycytidine), we observed an increase in GPRC5A protein abundance. On the other hand, when we knocked down GPRC5A we sensitized pancreatic cancer cells to gemcitabine. Through further experimentation we showed that the monotonic increase in GPRC5A protein levels that we observe for the first 18 h following gemcitabine treatment results from interactions between GPRC5A's mRNA and the RNA-binding protein HuR, which is an established key mediator of gemcitabine's efficacy in cancer cells. As we discovered, the interaction between GPRC5A and HuR is mediated by at least one HuR-binding site in GPRC5A's mRNA. Our findings indicate that

  8. AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

    PubMed

    Raghavan, Cibin T; Nagaraj, Ram H

    2016-08-01

    Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis. PMID:27263094

  9. Contacts between pigmented retina epithelial cells in culture.

    PubMed

    Middleton, C A; Pegrum, S M

    1976-11-01

    The behaviour of primary cultures of dissociated embryonic chick pigmented retina epithelial (PRE) cells has been investigated. Isolated PRE cells have a mean speed of locomotion of 7-16 mum/h. Collisions between the cells normally result in the development of stable contacts between the cells involved. This leads to a gradual reduction in the number of isolated cells and an increase in the number of cells incorporated into islands. Ultrastructural observations of islands of cells after 24 h in culture show that junctional complexes are present between the cells. These complexes consist of 2 components: (a) an apically situated region of focal tight junctions and/or gap junctions, and (b) a more ventrally located zonula adhaerens with associated cytoplasmic filaments forming a band running completely around the periphery of each cell. The intermembrane gap in the region of the zonula is 6-0-12-0 nm. The junctional complexes become more differentiated with time and after 48 h in culture consist of an extensive region of tight junctions and/or gap junctions and a more specialized zonula adhaerens. It is suggested that the development of junctional complexes may be responsible for the stable contacts that the cells display in culture.

  10. Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells.

    PubMed

    Chen, Xiuping; Guo, Feiye; LeBlanc, Michelle E; Ding, Ying; Zhang, Chenming; Shakya, Akhalesh; Li, Wei

    2016-08-01

    Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance.

  11. Barrier Epithelial Cells and the Control of Type 2 Immunity.

    PubMed

    Hammad, Hamida; Lambrecht, Bart N

    2015-07-21

    Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease.

  12. Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

    PubMed

    Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

    2016-05-01

    Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.

  13. Expression of tracheal antimicrobial peptide in bovine mammary epithelial cells.

    PubMed

    López-Meza, Joel E; Gutiérrez-Barroso, Angelina; Ochoa-Zarzosa, Alejandra

    2009-08-01

    The production of antimicrobial peptides is an important key of innate immunity. Tracheal antimicrobial peptide (TAP) expression has been reported in bovine tracheal epithelial cells and it can be modulated by bacterial infection or bacterial components. In mammary gland TAP expression has been reported, but the cell type that produces it is unknown. The objective of this work was to evaluate if bovine mammary epithelial cells (bMEC) express TAP mRNA, and evaluate the regulation of its expression in response to Staphylococcus aureus infection, bovine prolactin (bPRL) or acetyl salicylic acid (ASA). By retrotranscription and PCR, we demonstrated that bMEC express TAP mRNA. bMEC infected with live S. aureus down-regulates TAP expression, whereas the challenge with gentamicin-killed S. aureus up-regulates it. Also, bPRL do not significantly modify TAP expression, but in the presence of 5 mM ASA it was down-regulated, suggesting that nuclear factor kappaB (NF-kappaB) pathway can be involved in its regulation. PMID:19181355

  14. Proteomic analysis of nasal epithelial cells from cystic fibrosis patients.

    PubMed

    Jeanson, Ludovic; Guerrera, Ida Chiara; Papon, Jean-François; Chhuon, Cerina; Zadigue, Patricia; Prulière-Escabasse, Virginie; Amselem, Serge; Escudier, Estelle; Coste, André; Edelman, Aleksander

    2014-01-01

    The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.

  15. Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells.

    PubMed

    Ogunwobi, Olorunseun Olatunji; Beales, Ian L P

    2006-05-15

    Adiponectin is a recently described mediator secreted by adipose tissue. Here we report the growth promoting and pro-inflammatory actions of adiponectin on colonic epithelial cancer cells. Full-length and globular adiponectin produced an identical stimulation of HT-29 cell growth that was blocked by inhibition of adenylate cyclase and protein kinase A and partially inhibited by a pan-specific protein kinase C inhibitor, but was unaffected by specific inhibition of extracellular signal-related kinase (ERK) or p38 MAP kinase. Globular adiponectin but not full-length adiponectin significantly increased the secretion and mRNA levels of IL-8, GM-CSF and MCP-1. Globular adiponectin doubled IL-1beta-stimulated IL-8 and GM-CSF secretion. Adiponectin-stimulated cytokine secretion was blocked by pharmacological inhibitors of NF-kappaB, ERK and p38 MAP kinase. Globular adiponectin increased phosphorylation of both ERK and p38 MAP kinase and increased the nuclear translocation of active NF-kappaB. Adiponectin has pro-proliferative and pro-inflammatory actions on colonic epithelial cells; these appear to be differentially activated by the adiponectin isoforms. Adiponectin may have a role in the regulation of gastrointestinal mucosal function, inflammation and colon carcinogenesis.

  16. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    PubMed Central

    2009-01-01

    Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide. PMID:19650888

  17. Specific parasitism of purified vaginal epithelial cells by Trichomonas vaginalis.

    PubMed Central

    Alderete, J F; Demeś, P; Gombosova, A; Valent, M; Fabusová, M; Jánoska, A; Stefanovic, J; Arroyo, R

    1988-01-01

    Human vaginal epithelial cells (VECs) from vaginal swabs obtained from normal women or from patients with trichomoniasis were purified, and VEC parasitism by Trichomonas vaginalis was examined. Trichomonads bound equally well to live or dead VECs, and up to 20% of VECs were parasitized. Trichomonal cytadherence of human VECs was time, temperature, and pH dependent. Saturation binding levels of live trichomonads to VECs gave approximately 2 organisms adherent to parasitized VEC. No differences in cytadherence levels were detected by different isolates to VECs from the same patient compared with adherence to VECs from normal individuals. Trypsinized, live T. vaginalis organisms failed to recognize VECs. A ligand assay identified four adhesin candidates, and only organisms without a prominent immunogen on the surface (negative phenotype) cytadhered to VECs and synthesized the adhesins, confirming the results of a recently published report by us on adherence to HeLa cell monolayers (J. F. Alderete and G. E. Garza, Infect. Immun. 56:28-33, 1988). These data show the ability of T. vaginalis to parasitize human vaginal epithelial cells in a specific receptor-ligand manner. PMID:3262088

  18. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells.

    PubMed

    Huang, Minghai; Wang, Bowen; Wan, Pengxia; Liang, Xuanwei; Wang, Xiaoran; Liu, Ying; Zhou, Qiang; Wang, Zhichong

    2015-02-01

    Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.

  19. K+ and Na+ absorption by outer sulcus epithelial cells.

    PubMed

    Marcus, D C; Chiba, T

    1999-08-01

    Transduction of sound into nerve impulses by hair cells depends on modulation of a current carried primarily by K+ into the cell across apical transduction channels that are permeable to cations. The cochlear function thus depends on active secretion of K+ accompanied by absorption of Na+ by epithelial cells enclosing the cochlear duct. The para-sensory cells which participate in the absorption of Na+ (down to the uniquely low level of 1 mM) were previously unidentified and the existence of a para-sensory pathway which actively absorbs K+ was previously unknown. A relative short circuit current (Isc,probe, measured as the extracellular current density with a vibrating electrode) was directed into the apical side of the outer sulcus epithelium, decreased by ouabain (1 mM), an inhibitor of Na+, K(+)-ATPase, and found to depend on bath Na+ and K+ but on neither Ca2+ nor Cl-. Isc,probe was shown to be an active current by its sensitivity to ouabain. On-cell patch clamp recordings of the apical membrane of outer sulcus cells displayed a channel activity, which carried inward currents under conditions identical to those used to measure Isc,probe. Both Isc,probe and non-selective cation channels (27.4+/-0.6 ps, n = 22) in excised outside-out patches from the apical membrane were inhibited by Gd3+ (1 mM). Ics,prob was also inhibited by 5 mM lidocaine, 1 mM quinine and 500 microM amiloride but not by 10 microM amiloride. These results demonstrate that outer sulcus epithelial cells contribute to the homeostasis of endolymph by actively absorbing Na+ and K+. An entry pathway in the apical membrane was shown to be through non-selective cation channels that were sensitive to Gd3+.

  20. Rac1 mediates intestinal epithelial cell apoptosis via JNK.

    PubMed

    Jin, Shi; Ray, Ramesh M; Johnson, Leonard R

    2006-12-01

    Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of the intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The present study tested the hypothesis that Rac1 mediates TNF-alpha-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-alpha-induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of constitutively active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC-23766 (Rac1 inhibitor) or expression of dominant negative Rac1 protects cells from TNF-alpha-induced apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 before the administration of apoptotic stimuli significantly prevents TNF-alpha-induced activation of JNK1/2, the key proapoptotic regulator in IEC-6 cells. Inhibition of Rac1 does not modulate TNF-alpha-induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity by U-0126 and LY-294002, respectively, increased TNF-alpha-induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC-23766 alone in response to TNF-alpha. Thus, Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during TNF-alpha-induced apoptosis. Although p38 MAPK is activated in response to TNF-alpha, inhibition of p38 MAPK did not decrease apoptosis. Rac1 inhibition did not alter p38 MAPK activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in proapoptotic pathways in intestinal epithelial cells.

  1. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    PubMed

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency. PMID:26673160

  2. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    PubMed

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

  3. Hertwig's epithelial root sheath cell behavior during initial acellular cementogenesis in rat molars.

    PubMed

    Yamamoto, Tsuneyuki; Yamamoto, Tomomaya; Yamada, Tamaki; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

    2014-11-01

    This study was designed to examine developing acellular cementum in rat molars by immunohistochemistry, to elucidate (1) how Hertwig's epithelial root sheath disintegrates and (2) whether epithelial sheath cells transform into cementoblasts through epithelial-mesenchymal transition (EMT). Initial acellular cementogenesis was divided into three developmental stages, which can be seen in three different portions of the root: portion 1, where the epithelial sheath is intact; portion 2, where the epithelial sheath becomes fragmented; and portion 3, where acellular cementogenesis begins. Antibodies against three kinds of matrix proteinases, which degrade epithelial sheath-maintaining factors, including basement membrane and desmosomes, were used to investigate proteolytic activity of the epithelial sheath. Tissue non-specific alkaline phosphatase (TNALP) and keratin were used to investigate EMT. Epithelial sheath cells showed immunoreactivity for all three enzymes at fragmentation, which suggests that epithelial sheath disintegration is enzymatically mediated. Dental follicle cells and cementoblasts showed intense immunoreactivity for TNALP, and from portion 1 through to 3, the reaction extended from the alveolar bone-related zone to the root-related zone. Cells possessing keratin/TNALP double immunoreactivity were virtually absent. Keratin-positive epithelial sheath cells showed negligible immunoreactivity for TNALP, and epithelial cells did not appear to migrate to the dental follicle. Together, these findings suggest that a transition phenotype between epithelial cells and cementoblasts does not exist in the developing dental follicle and hence that epithelial sheath cells do not undergo EMT during initial acellular cementogenesis. In brief, this study supports the notion that cementoblasts derive from the dental follicle. PMID:24859538

  4. Production of arachidonic and linoleic acid metabolites by guinea pig tracheal epithelial cells

    SciTech Connect

    Oosthuizen, M.J.; Engels, F.; Van Esch, B.; Henricks, P.A.; Nijkamp, F.P. )

    1990-08-01

    Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.

  5. OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine both in vitro and in vivo.

    PubMed

    Zhi, Xiao; Chen, Wei; Xue, Fei; Liang, Chao; Chen, Bryan Wei; Zhou, Yue; Wen, Liang; Hu, Liqiang; Shen, Jian; Bai, Xueli; Liang, Tingbo

    2015-09-22

    Despite its relative rarity, pancreatic ductal adenocarcinoma (PDAC) accounts for a large percentage of cancer deaths. In this study, we investigated the in vitro efficacy of OSI-027, a selective inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, to treat PDAC cell lines alone, and in combination with gemcitabine (GEM). Similarly, we tested the efficacy of these two compounds in a xenograft mouse model of PDAC. OSI-027 significantly arrested cell cycle in G0/G1 phase, inhibited the proliferation of Panc-1, BxPC-3, and CFPAC-1 cells, and downregulated mTORC1, mTORC2, phospho-Akt, phospho-p70S6K, phospho-4E-BP1, cyclin D1, and cyclin-dependent kinase 4 (CDK4) in these cells. Moreover, OSI-027 also downregulated multidrug resistance (MDR)-1, which has been implicated in chemotherapy resistance in PDAC cells and enhanced apoptosis induced by GEM in the three PDAC cell lines. When combined, OSI-027 with GEM showed synergistic cytotoxic effects both in vitro and in vivo. This is the first evidence of the efficacy of OSI-027 in PDAC and may provide the groundwork for a new clinical PDAC therapy. PMID:26213847

  6. Growth of respiratory syncytial virus in mink lung epithelial cells.

    PubMed

    Yeolekar, L R; Damle, R G; Basu, A; Rao, B L

    2002-12-01

    Mink lung epithelial cells (Mv-1-Lu) were tested for their ability to support the growth and serial passage of respiratory syncytial virus (RSV) in vitro. Indian isolates of RSV induced distinctive cytopathic effect with typical rounding of cells followed by detachment with more than 50 per cent cells showing bright fluorescence using anti-RSV monoclonal antibodies in immunofluorescence test. Serial passage of RSV was possible in Mv-1-Lu cells without loss of sensitivity of the cells for virus growth. Titration of cell associated virus and virus released in the supernatant indicated that 60 per cent of the virus was released in the supernatant, and 40 per cent remained cell associated. Transmission electron microscopic studies of negatively stained RSV particles and ultra-thin sections of RSV infected Mv-1-Lu cells showed roughly spherical particles with club shaped projections, budding from the cytoplasmic membrane. These results indicate that Mv-1-Lu cell line is suitable for the growth and propagation of RSV.

  7. Effect of curcumin on aging retinal pigment epithelial cells.

    PubMed

    Zhu, Wei; Wu, Yan; Meng, Yi-Fang; Wang, Jin-Yu; Xu, Ming; Tao, Jian-Jun; Lu, Jiong

    2015-01-01

    Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE) cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 µM, 40 µM, and 80 µM. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD.

  8. Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells.

    PubMed

    Millaku, Agron; Drobne, Damjana; Torkar, Matjaz; Novak, Sara; Remškar, Maja; Pipan-Tkalec, Živa

    2013-09-15

    We provide data obtained by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) on the interaction of ingested tungsten nanofibers with epithelial cells of the digestive tubes of a test organism Porcellio scaber. Conventional toxicity endpoints including feeding behaviour, weight loss and mortality were also measured in each investigated animal. No toxicity was detected in any of exposed animals after 14 days of feeding on tungsten nanofiber dosed food, but when nanofibers enter the digestive system they can react with epithelial cells of the digestive tubes, becoming physically inserted into the cells. In this way, nanofibers can injure the epithelial cells of digestive gland tubes when they are ingested with food. Our SEM data suggest that peristaltic forces may have an important role, not predicted by in vitro experiments, in the interactions of nanomaterials with digestive intestinal cells.

  9. Equine tracheal epithelial membrane strips - An alternate method for examining epithelial cell arachidonic acid metabolism

    SciTech Connect

    Gray, P.R.; Derksen, F.J.; Robinson, N.E.; Peter-Golden, M.L. Univ. of Michigan, Ann Arbor )

    1990-02-26

    Arachidonic acid metabolism by tracheal epithelium can be studied using enzymatically dispersed cell suspensions or cell cultures. Both techniques require considerable tissue disruption and manipulation and may not accurately represent in vivo activity. The authors have developed an alternate method for obtaining strips of equine tracheal epithelium without enzymatic digestion. In the horse, a prominent elastic lamina supports the tracheal epithelium. By physical splitting this lamina, they obtained strips ({le}12 x 1.5 cm) of pseudostratified columnar epithelium attached to a layer of elastic tissue 30-100 {mu}m thick. Epithelial strips (1.2 x 0.5 cm) were attached to plexiglass rods and incubated with ({sup 3}H)arachidonic acid in M199 medium (0.5 {mu}Ci/ml) for 24 hours at 37C. The strips incorporated 36{+-}4% (mean {+-} SEM) of the total radioactivity and released 8.0{+-}1.2% of incorporated radioactivity when stimulated by 5.0 {mu}M calcium ionophore A23187. The extracted supernatant was processed using HPLC, resulting in peaks of radioactivity that co-eluted with authentic PGE{sub 2}, PGF{sub 2}{alpha}, and 12-HETE standards. The greatest activity corresponded to the PGE{sub 2} and PGF{sub 2}{alpha} standards, which is a similar pattern to that reported for cultured human tracheal epithelium.

  10. Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development.

    PubMed

    Klug, D B; Crouch, E; Carter, C; Coghlan, L; Conti, C J; Richie, E R

    2000-02-15

    We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.

  11. MicroRNA-15a/16 Regulates Apoptosis of Lung Epithelial Cells After Oxidative Stress

    PubMed Central

    Cao, Yong; Zhang, Duo; Moon, Hyung-Geun; Lee, Heedoo; Haspel, Jeffrey A; Hu, Kebin; Xie, Lixin; Jin, Yang

    2016-01-01

    Lung epithelial cell apoptosis is an important feature of hyperoxia-induced lung injury. The death receptor–associated extrinsic pathway and mitochondria-associated intrinsic pathway both mediate the development of lung epithelial cell apoptosis. Despite decades of research, molecular mechanisms of hyperoxia-induced epithelial cell apoptosis remain incompletely understood. Here, we report a novel regulatory paradigm in response to hyperoxia-associated oxidative stress. Hyperoxia markedly upregulated microRNA (miR)-15a/16 levels in lung epithelial cells, bronchoalveolar lavage fluid (BALF) and lung tissue. This effect was mediated by hyperoxia-induced reactive oxygen species. Functionally, miR-15a/16 inhibitors induced caspase-3–mediated lung epithelial cell apoptosis, in the presence of hyperoxia. MiR-15a/16 inhibitors robustly enhanced FADD level and downregulated Bcl-2 expression. Consistently, cleaved caspase-8 and -9 were highly induced in the miR-15a/16–deficient cells, after hyperoxia. Using airway epithelial cell–specific, miR-15a/16–/– mice, we found that Bcl-2 was significantly reduced in lung epithelial cells in vivo after hyperoxia. In contrast, caspase-3, caspase-8 and Bcl-2–associated death promoter (BAD) were highly elevated in the miR-15a/16–/– epithelial cells in vivo. Interestingly, in lung epithelial malignant cells, rather than benign cells, deletion of miR-15a/16 prevented apoptosis. Furthermore, deletion of miR-15a/16 in macrophages also prohibited apoptosis, which is the opposite of what we have found in normal lung epithelial cells. Taken together, our data suggested that miR-15a/16 may exert differential roles in different cell types. MiR-15a/16 deficiency results in lung epithelial cell apoptosis in response to hyperoxia, via modulating both intrinsic and extrinsic apoptosis pathways. PMID:27257854

  12. NK cells are necessary for recovery of corneal CD11c+ dendritic cells after epithelial abrasion injury

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mechanisms controlling CD11c(+) MHCII(+) DCs during corneal epithelial wound healing were investigated in a murine model of corneal abrasion. Selective depletion of NKp46(+) CD3- NK cells that normally migrate into the cornea after epithelial abrasion resulted in >85% reduction of the epithelial CD1...

  13. Three-Dimensional Cultures of Mouse Mammary Epithelial Cells

    PubMed Central

    Mroue, Rana; Bissell, Mina J.

    2013-01-01

    The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our

  14. Potassium currents in rat type II alveolar epithelial cells.

    PubMed Central

    DeCoursey, T E; Jacobs, E R; Silver, M R

    1988-01-01

    1. Type II alveolar epithelial cells isolated from adult rats and grown in primary culture were studied using the whole-cell configuration of the gigohm-seal voltage clamp technique. 2. The average specific capacitance of type II cells was 2.5 microF/cm2, suggesting that type II cell membranes in vitro are irregular, with an actual area more than twice the apparent area. 3. Most type II cells have time- and voltage-dependent outward currents carried by potassium ions. Potassium currents activate with a sigmoid time course upon membrane depolarization, and inactivate during maintained depolarization. The average maximum whole-cell K+ conductance was 1.6 nS. 4. Two distinct types of K+-selective channels underlie outward currents in type II cells. Most cells have currents resembling delayed rectifier K+ currents in skeletal muscle, nerve and immune cells. A few cells had a different type of K+ conductance which is more sensitive to block by tetraethylammonium ions, has faster 'tail currents', and activates at more positive potentials. 5. In some experiments, individual type II cells were identified by staining with phosphine, a fluorescent dye which is concentrated in lamellar bodies. Both types of K+ channels were seen in type II cells identified with this dye. 6. Phosphine added to the bathing solution reversibly reduced K+ currents and shifted K+ channel activation to more positive potentials. Excitation of phosphine to fluoresce reduced irreversibly K+ currents in type II cells. The usefulness of phosphine as a means of identifying cells for study is discussed. PMID:2457683

  15. The co-factor of LIM domains (CLIM/LDB/NLI) maintains basal mammary epithelial stem cells and promotes breast tumorigenesis.

    PubMed

    Salmans, Michael L; Yu, Zhengquan; Watanabe, Kazuhide; Cam, Eric; Sun, Peng; Smyth, Padhraic; Dai, Xing; Andersen, Bogi

    2014-07-01

    Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans.

  16. Proteomic and Bioinformatic Profile of Primary Human Oral Epithelial Cells

    PubMed Central

    Ghosh, Santosh K.; Yohannes, Elizabeth; Bebek, Gurkan; Weinberg, Aaron; Jiang, Bin; Willard, Belinda; Chance, Mark R.; Kinter, Michael T.; McCormick, Thomas S.

    2012-01-01

    Wounding of the oral mucosa occurs frequently in a highly septic environment. Remarkably, these wounds heal quickly and the oral cavity, for the most part, remains healthy. Deciphering the normal human oral epithelial cell (NHOEC) proteome is critical for understanding the mechanism(s) of protection elicited when the mucosal barrier is intact, as well as when it is breached. Combining 2D gel electrophoresis with shotgun proteomics resulted in identification of 1662 NHOEC proteins. Proteome annotations were performed based on protein classes, molecular functions, disease association and membership in canonical and metabolic signaling pathways. Comparing the NHOEC proteome with a database of innate immunity-relevant interactions (InnateDB) identified 64 common proteins associated with innate immunity. Comparison with published salivary proteomes revealed that 738/1662 NHOEC proteins were common, suggesting that significant numbers of salivary proteins are of epithelial origin. Gene ontology analysis showed similarities in the distributions of NHOEC and saliva proteomes with regard to biological processes, and molecular functions. We also assessed the inter-individual variability of the NHOEC proteome and observed it to be comparable with other primary cells. The baseline proteome described in this study should serve as a resource for proteome studies of the oral mucosa, especially in relation to disease processes. PMID:23035736

  17. The PCP pathway regulates Baz planar distribution in epithelial cells.

    PubMed

    Aigouy, Benoit; Le Bivic, André

    2016-01-01

    The localisation of apico-basal polarity proteins along the Z-axis of epithelial cells is well understood while their distribution in the plane of the epithelium is poorly characterised. Here we provide a systematic description of the planar localisation of apico-basal polarity proteins in the Drosophila ommatidial epithelium. We show that the adherens junction proteins Shotgun and Armadillo, as well as the baso-lateral complexes, are bilateral, i.e. present on both sides of cell interfaces. In contrast, we report that other key adherens junction proteins, Bazooka and the myosin regulatory light chain (Spaghetti squash) are unilateral, i.e. present on one side of cell interfaces. Furthermore, we demonstrate that planar cell polarity (PCP) and not the apical determinants Crumbs and Par-6 control Bazooka unilaterality in cone cells. Altogether, our work unravels an unexpected organisation and combination of apico-basal, cytoskeletal and planar polarity proteins that is different on either side of cell-cell interfaces and unique for the different contacts of the same cell. PMID:27624969

  18. The PCP pathway regulates Baz planar distribution in epithelial cells

    PubMed Central

    Aigouy, Benoit; Le Bivic, André

    2016-01-01

    The localisation of apico-basal polarity proteins along the Z-axis of epithelial cells is well understood while their distribution in the plane of the epithelium is poorly characterised. Here we provide a systematic description of the planar localisation of apico-basal polarity proteins in the Drosophila ommatidial epithelium. We show that the adherens junction proteins Shotgun and Armadillo, as well as the baso-lateral complexes, are bilateral, i.e. present on both sides of cell interfaces. In contrast, we report that other key adherens junction proteins, Bazooka and the myosin regulatory light chain (Spaghetti squash) are unilateral, i.e. present on one side of cell interfaces. Furthermore, we demonstrate that planar cell polarity (PCP) and not the apical determinants Crumbs and Par-6 control Bazooka unilaterality in cone cells. Altogether, our work unravels an unexpected organisation and combination of apico-basal, cytoskeletal and planar polarity proteins that is different on either side of cell-cell interfaces and unique for the different contacts of the same cell. PMID:27624969

  19. Epithelial mechanobiology, skin wound healing, and the stem cell niche.

    PubMed

    Evans, Nicholas D; Oreffo, Richard O C; Healy, Eugene; Thurner, Philipp J; Man, Yu Hin

    2013-12-01

    Skin wound healing is a vital process that is important for re-establishing the epithelial barrier following disease or injury. Aberrant or delayed skin wound healing increases the risk of infection, causes patient morbidity, and may lead to the formation of scar tissue. One of the most important events in wound healing is coverage of the wound with a new epithelial layer. This occurs when keratinocytes at the wound periphery divide and migrate to re-populate the wound bed. Many approaches are under investigation to promote and expedite this process, including the topical application of growth factors and the addition of autologous and allogeneic tissue or cell grafts. The mechanical environment of the wound site is also of fundamental importance for the rate and quality of wound healing. It is known that mechanical stress can influence wound healing by affecting the behaviour of cells within the dermis, but it remains unclear how mechanical forces affect the healing epidermis. Tensile forces are known to affect the behaviour of cells within epithelia, however, and the material properties of extracellular matrices, such as substrate stiffness, have been shown to affect the morphology, proliferation, differentiation and migration of many different cell types. In this review we will introduce the structure of the skin and the process of wound healing. We will then discuss the evidence for the effect of tissue mechanics in re-epithelialisation and, in particular, on stem cell behaviour in the wound microenvironment and in intact skin. We will discuss how the elasticity, mechanical heterogeneity and topography of the wound extracellular matrix impact the rate and quality of wound healing, and how we may exploit this knowledge to expedite wound healing and mitigate scarring. PMID:23746929

  20. Epithelial mechanobiology, skin wound healing, and the stem cell niche.

    PubMed

    Evans, Nicholas D; Oreffo, Richard O C; Healy, Eugene; Thurner, Philipp J; Man, Yu Hin

    2013-12-01

    Skin wound healing is a vital process that is important for re-establishing the epithelial barrier following disease or injury. Aberrant or delayed skin wound healing increases the risk of infection, causes patient morbidity, and may lead to the formation of scar tissue. One of the most important events in wound healing is coverage of the wound with a new epithelial layer. This occurs when keratinocytes at the wound periphery divide and migrate to re-populate the wound bed. Many approaches are under investigation to promote and expedite this process, including the topical application of growth factors and the addition of autologous and allogeneic tissue or cell grafts. The mechanical environment of the wound site is also of fundamental importance for the rate and quality of wound healing. It is known that mechanical stress can influence wound healing by affecting the behaviour of cells within the dermis, but it remains unclear how mechanical forces affect the healing epidermis. Tensile forces are known to affect the behaviour of cells within epithelia, however, and the material properties of extracellular matrices, such as substrate stiffness, have been shown to affect the morphology, proliferation, differentiation and migration of many different cell types. In this review we will introduce the structure of the skin and the process of wound healing. We will then discuss the evidence for the effect of tissue mechanics in re-epithelialisation and, in particular, on stem cell behaviour in the wound microenvironment and in intact skin. We will discuss how the elasticity, mechanical heterogeneity and topography of the wound extracellular matrix impact the rate and quality of wound healing, and how we may exploit this knowledge to expedite wound healing and mitigate scarring.

  1. Hepatocyte-Ductal Transdifferentiation Is Mediated by Reciprocal Repression of SOX9 and C/EBPα

    PubMed Central

    O'Neill, Kathy E.; Thowfeequ, Shifaan; Li, Wan-Chun; Eberhard, Daniel; Dutton, James R.; Slack, Jonathan M.W.

    2014-01-01

    Abstract Primary hepatocytes rapidly dedifferentiate when cultured in vitro. We have studied the mechanism of hepatocyte dedifferentiation by using two culture media: one that maintains hepatocytes in a differentiated state and another that allows dedifferentiation. We show that dedifferentiation involves partial transformation of hepatocytes into cells that resemble biliary epithelial cells. Lineage labeling and time-lapse filming confirm that the dedifferentiated cells are derived from hepatocytes and not from contaminating ductal or fibroblastic cells in the original culture. Furthermore, we establish that the conversion of hepatocytes to biliary-like cells is regulated by mutual antagonism of CCAAT/enhancer binding protein alpha (C/EBPα) and SOX9, which have opposing effects on the expression of hepatocyte and ductal genes. Thus, hepatocyte dedifferentiation induces the biliary gene expression program by alleviating C/EBPα-mediated repression of Sox9. We propose that reciprocal antagonism of C/EBPα and SOX9 also operates in the formation of hepatocytes and biliary ducts from hepatoblasts during normal embryonic development. These data demonstrate that reprogramming of differentiated cells can be used to model the acquisition and maintenance of cell fate in vivo. PMID:25153359

  2. Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions.

    PubMed

    Short, Kirsty R; Kasper, Jennifer; van der Aa, Stijn; Andeweg, Arno C; Zaaraoui-Boutahar, Fatiha; Goeijenbier, Marco; Richard, Mathilde; Herold, Susanne; Becker, Christin; Scott, Dana P; Limpens, Ronald W A L; Koster, Abraham J; Bárcena, Montserrat; Fouchier, Ron A M; Kirkpatrick, Charles James; Kuiken, Thijs

    2016-03-01

    A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection.

  3. Human Epithelial Cells Discriminate between Commensal and Pathogenic Interactions with Candida albicans.

    PubMed

    Rast, Timothy J; Kullas, Amy L; Southern, Peter J; Davis, Dana A

    2016-01-01

    The commensal fungus, Candida albicans, can cause life-threatening infections in at risk individuals. C. albicans colonizes mucosal surfaces of most people, adhering to and interacting with epithelial cells. At low concentrations, C. albicans is not pathogenic nor does it cause epithelial cell damage in vitro; at high concentrations, C. albicans causes mucosal infections and kills epithelial cells in vitro. Here we show that while there are quantitative dose-dependent differences in exposed epithelial cell populations, these reflect a fundamental qualitative difference in host cell response to C. albicans. Using transcriptional profiling experiments and real time PCR, we found that wild-type C. albicans induce dose-dependent responses from a FaDu epithelial cell line. However, real time PCR and Western blot analysis using a high dose of various C. albicans strains demonstrated that these dose-dependent responses are associated with ability to promote host cell damage. Our studies support the idea that epithelial cells play a key role in the immune system by monitoring the microbial community at mucosal surfaces and initiating defensive responses when this community is dysfunctional. This places epithelial cells at a pivotal position in the interaction with C. albicans as epithelial cells themselves promote C. albicans stimulated damage.

  4. Human Epithelial Cells Discriminate between Commensal and Pathogenic Interactions with Candida albicans

    PubMed Central

    Rast, Timothy J.; Kullas, Amy L.; Southern, Peter J.; Davis, Dana A.

    2016-01-01

    The commensal fungus, Candida albicans, can cause life-threatening infections in at risk individuals. C. albicans colonizes mucosal surfaces of most people, adhering to and interacting with epithelial cells. At low concentrations, C. albicans is not pathogenic nor does it cause epithelial cell damage in vitro; at high concentrations, C. albicans causes mucosal infections and kills epithelial cells in vitro. Here we show that while there are quantitative dose-dependent differences in exposed epithelial cell populations, these reflect a fundamental qualitative difference in host cell response to C. albicans. Using transcriptional profiling experiments and real time PCR, we found that wild-type C. albicans induce dose-dependent responses from a FaDu epithelial cell line. However, real time PCR and Western blot analysis using a high dose of various C. albicans strains demonstrated that these dose-dependent responses are associated with ability to promote host cell damage. Our studies support the idea that epithelial cells play a key role in the immune system by monitoring the microbial community at mucosal surfaces and initiating defensive responses when this community is dysfunctional. This places epithelial cells at a pivotal position in the interaction with C. albicans as epithelial cells themselves promote C. albicans stimulated damage. PMID:27088599

  5. Human Epithelial Cells Discriminate between Commensal and Pathogenic Interactions with Candida albicans.

    PubMed

    Rast, Timothy J; Kullas, Amy L; Southern, Peter J; Davis, Dana A

    2016-01-01

    The commensal fungus, Candida albicans, can cause life-threatening infections in at risk individuals. C. albicans colonizes mucosal surfaces of most people, adhering to and interacting with epithelial cells. At low concentrations, C. albicans is not pathogenic nor does it cause epithelial cell damage in vitro; at high concentrations, C. albicans causes mucosal infections and kills epithelial cells in vitro. Here we show that while there are quantitative dose-dependent differences in exposed epithelial cell populations, these reflect a fundamental qualitative difference in host cell response to C. albicans. Using transcriptional profiling experiments and real time PCR, we found that wild-type C. albicans induce dose-dependent responses from a FaDu epithelial cell line. However, real time PCR and Western blot analysis using a high dose of various C. albicans strains demonstrated that these dose-dependent responses are associated with ability to promote host cell damage. Our studies support the idea that epithelial cells play a key role in the immune system by monitoring the microbial community at mucosal surfaces and initiating defensive responses when this community is dysfunctional. This places epithelial cells at a pivotal position in the interaction with C. albicans as epithelial cells themselves promote C. albicans stimulated damage. PMID:27088599

  6. Ferritinophagy drives uropathogenic Escherichia coli persistence in bladder epithelial cells.

    PubMed

    Bauckman, Kyle A; Mysorekar, Indira U

    2016-05-01

    Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.

  7. Contact inhibition of phagocytosis in epithelial sheets: alterations of cell surface properties induced by cell-cell contacts.

    PubMed Central

    Vasiliev, J M; Gelfand, I M; Domnina, L V; Zacharova, O S; Ljubimov, A V

    1975-01-01

    Contact inhibition of phagocytosis was found to be characteristic for epithelial sheets formed in cultures by several cell types: normal and transformed mouse kidney cells, and differentiated mouse hepatoma cells. In these sheets most central cells surrounded by other cells had very low phagocytic activity. In contrast, marginal cells having a free edge were able to perform an active phagocytosis. Contact inhibition of phagocytosis was absent in dense cultures of mouse embryo fibroblasts and in cultures of anaplastic mouse hepatoma 22a. The upper surface of epithelial sheets was nonadhesive for prelabeled epithelial cells and fibroblasts. In contrast, the upper surface of dense cultures of mouse fibroblasts was adhesive for these cells. These and other data strengthen the suggestion that contact inhibition of phagocytosis is a result of different adhesiveness of the upper cell surface and of the surfaces near the free edge. Agents inhibiting cell surface movements at the free edges of marginal epithelial cells (cytochalasin, azide, sorbitol, low temperature) prevented adhesion of particles to these edges. Possibly, the surface of actively moving cytoplasmic processes is the only cell part that has adhesive properties necessary for the formation of attachments with other cellular and noncellular surfaces. In epithelial sheets, in contrast to fibroblast cultures, Colcemid did not activate movements of immobile contacting cell edges. These results indicate that mechanisms of contact immobilization of cell surface may be different in epithelium and fibroblasts. Firm contacts formed between epithelial cells are sufficient for stable immobilization of the surface; additional stabilization of the surface by microtubules is not essential. Fibroblasts do not form firm contacts and the Colcemid-sensitive stabilization process is essential for maintenance of the immobile state of their surfaces. Differences in the stability of cell surface immobilization produced by cell-cell

  8. The yin and yang of intestinal epithelial cells in controlling dendritic cell function

    PubMed Central

    Iliev, Iliyan D.; Matteoli, Gianluca; Rescigno, Maria

    2007-01-01

    Recent work suggests that dendritic cells (DCs) in mucosal tissues are “educated” by intestinal epithelial cells (IECs) to suppress inflammation and promote immunological tolerance. After attack by pathogenic microorganisms, however, “non-educated” DCs are recruited from nearby areas, such as the dome of Peyer's patches (PPs) and the blood, to initiate inflammation and the ensuing immune response to the invader. Differential epithelial cell (EC) responses to commensals and pathogens may control these two tolorogenic and immunogenic functions of DCs. PMID:17893197

  9. Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

    PubMed

    Schwagerus, Elena; Sladek, Svenja; Buckley, Stephen T; Armas-Capote, Natalia; Alvarez de la Rosa, Diego; Harvey, Brian J; Fischer, Horst; Illek, Beate; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

    2015-11-01

    Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

  10. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  11. Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells

    PubMed Central

    Karuri, Nancy W.; Liliensiek, Sara; Teixeira, Ana I.; Abrams, George; Campbell, Sean; Nealey, Paul F.; Murphy, Christopher J.

    2006-01-01

    Summary The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design. PMID:15226393

  12. Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells.

    PubMed

    Karuri, Nancy W; Liliensiek, Sara; Teixeira, Ana I; Abrams, George; Campbell, Sean; Nealey, Paul F; Murphy, Christopher J

    2004-07-01

    The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design.

  13. Control of cell mechanics by RhoA and calcium fluxes during epithelial scattering.

    PubMed

    Haws, Hillary J; McNeil, Melissa A; Hansen, Marc D H

    2016-01-01

    Epithelial tissues use adherens junctions to maintain tight interactions and coordinate cellular activities. Adherens junctions are remodeled during epithelial morphogenesis, including instances of epithelial-mesenchymal transition, or EMT, wherein individual cells detach from the tissue and migrate as individual cells. EMT has been recapitulated by growth factor induction of epithelial scattering in cell culture. In culture systems, cells undergo a highly reproducible series of cell morphology changes, most notably cell spreading followed by cellular compaction and cell migration. These morphology changes are accompanied by striking actin rearrangements. The current evidence suggests that global changes in actomyosin-based cellular contractility, first a loss of contractility during spreading and its activation during cell compaction, are the main drivers of epithelial scattering. In this review, we focus on how spreading and contractility might be controlled during epithelial scattering. While we propose a central role for RhoA, which is well known to control cellular contractility in multiple systems and whose role in epithelial scattering is well accepted, we suggest potential roles for additional cellular systems whose role in epithelial cell biology has been less well documented. In particular, we propose critical roles for vesicle recycling, calcium channels, and calcium-dependent kinases. PMID:27583192

  14. Differences in the adhesive properties of Neisseria meningitidis for human buccal epithelial cells and erythrocytes.

    PubMed Central

    Trust, T J; Gillespie, R M; Bhatti, A R; White, L A

    1983-01-01

    The ability of clinical and carrier isolates of Neisseria meningitidis to adhere to human buccal epithelial cells and erythrocytes was investigated. Four of the 10 fimbriated strains were able to hemagglutinate. Serial subculture of three of these strains resulted in a loss of ability to hemagglutinate and was coincident with a loss of fimbriation. Other fimbriated strains were unable to hemagglutinate but did adhere to buccal epithelial cells. Subculture of one of these strains for as many as 42 passages did not result in loss of fimbriation or ability to adhere to buccal epithelial cells. The attachment of this strain to buccal epithelial cells was inhibited by glycoconjugates. Further, pH exerted different influences on the attachment of hemagglutinating and non-hemagglutinating fimbriated strains to buccal epithelial cells and erythrocytes. The results suggest that different fimbrial mechanisms are involved in the attachment of N. meningitidis to different cell types and that hemagglutination is not an absolute test for fimbriae. PMID:6134676

  15. Recognition of Major Histocompatibility Complex Antigens on Cultured Human Biliary Epithelial Cells by Alloreactive Lymphocytes

    PubMed Central

    Saidman, Susan L.; Duquesnoy, Rene J.; Zeevi, Adriana; Fung, John J.; Starzl, Thomas E.; Demetris, A. Jake

    2010-01-01

    We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-γ-interferon monoclonal antibody. In addition, recombinant human γ-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell–induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody–blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system. PMID:1704868

  16. CAR regulates epithelial cell junction stability through control of E-cadherin trafficking

    PubMed Central

    Morton, Penny E.; Hicks, Alexander; Nastos, Theodoros; Santis, George; Parsons, Maddy

    2013-01-01

    CAR (Coxsackie and Adenovirus Receptor) is the primary docking receptor for typeB coxsackie viruses and subgroup C adenoviruses. CAR is a member of the JAM family of adhesion receptors and is located to both tight and adherens junctions between epithelial cells where it can assemble adhesive contacts through homodimerisation in trans. However, the role of CAR in controlling epithelial junction dynamics remains poorly understood. Here we demonstrate that levels of CAR in human epithelial cells play a key role in determining epithelial cell adhesion through control of E-cadherin stability at cell-cell junctions. Mechanistically, we show that CAR is phosphorylated within the C-terminus by PKCδ and that this in turn controls Src-dependent endocytosis of E-cadherin at cell junctions. This data demonstrates a novel role for CAR in regulating epithelial homeostasis. PMID:24096322

  17. Characterization of protamine uptake by opossum kidney epithelial cells.

    PubMed

    Nagai, Junya; Komeda, Takuji; Katagiri, Yuki; Yumoto, Ryoko; Takano, Mikihisa

    2013-01-01

    Protamine, a mixture of polypeptides that is rich in arginine, has been used clinically as an antidote to heparin overdoses and a complexing agent in a long-acting insulin preparation. When protamine is administered intravenously, its abundant accumulation in the kidneys has been reported. However, the renal uptake mechanism for protamine is not clear. In this study, we examined the transport mechanism for protamine in opossum kidney (OK) cells, a suitable in vitro model for renal proximal tubular epithelial cells. Flow cytometric analysis revealed that the association of fluorescein isothiocyanate (FITC)-labeled protamine from salmon (FITC-protamine) by OK cells was inhibited by unlabeled protamine in a concentration-dependent manner. The association of FITC-protamine was temperature- and energy-dependent. Confocal microscopy analysis showed that the fluorescence was localized in the cytoplasm and nucleus of OK cells. In addition, FITC-protamine association was inhibited by cationic drugs such as polycationic gentamicin and polymixin B, but it was increased by a basic amino acid, arginine. Inhibitors for clathrin- and caveolin-dependent endocytosis showed inhibitory effects on FITC-protamine association. Pretreatment with heparinase III partially but significantly decreased the association of FITC-protamine. These results suggest that protamine may be taken up by OK cells via receptor-mediated endocytosis, which may result in its localization in the cytoplasm and nucleus of the cells.

  18. Characterization of stress response in human retinal epithelial cells

    PubMed Central

    Giansanti, Vincenzo; Villalpando Rodriguez, Gloria E; Savoldelli, Michelle; Gioia, Roberta; Forlino, Antonella; Mazzini, Giuliano; Pennati, Marzia; Zaffaroni, Nadia; Scovassi, Anna Ivana; Torriglia, Alicia

    2013-01-01

    The pathogenesis of age-related macular degeneration (AMD) involves demise of the retinal pigment epithelium and death of photoreceptors. In this article, we investigated the response of human adult retinal pigmented epithelial (ARPE-19) cells to 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of Na+/H+ exchangers. We observed that ARPE-19 cells treated with HMA are unable to activate ‘classical’ apoptosis but they succeed to activate autophagy. In the first 2 hrs of HMA exposure, autophagy is efficient in protecting cells from death. Thereafter, autophagy is impaired, as indicated by p62 accumulation, and this protective mechanism becomes the executioner of cell death. This switch in autophagy property as a function of time for a single stimulus is here shown for the first time. The activation of autophagy was observed, at a lesser extent, with etoposide, suggesting that this event might be a general response of ARPE cells to stress and the most important pathway involved in cell resistance to adverse conditions and toxic stimuli. PMID:23205553

  19. Effect of Stratification on Surface Properties of Corneal Epithelial Cells

    PubMed Central

    Yáñez-Soto, Bernardo; Leonard, Brian C.; Raghunathan, Vijay Krishna; Abbott, Nicholas L.; Murphy, Christopher J.

    2015-01-01

    Purpose The purpose of this study was to determine the influence of mucin expression in an immortalized human corneal epithelial cell line (hTCEpi) on the surface properties of cells, such as wettability, contact angle, and surface heterogeneity. Methods hTCEpi cells were cultured to confluence in serum-free medium. The medium was then replaced by stratification medium to induce mucin biosynthesis. The mucin expression profile was analyzed using quantitative PCR and Western blotting. Contact angles were measured using a two-immiscible liquid method, and contact angle hysteresis was evaluated by tilting the apparatus and recording advancing and receding contact angles. The spatial distribution of mucins was evaluated with fluorescently labeled lectin. Results hTCEpi cells expressed the three main ocular mucins (MUC1, MUC4, and MUC16) with a maximum between days 1 and 3 of the stratification process. Upon stratification, cells caused a very significant increase in contact angle hysteresis, suggesting the development of spatially discrete and heterogeneously distributed surface features, defined by topography and/or chemical functionality. Although atomic force microscopy measurements showed no formation of appreciable topographic features on the surface of the cells, we observed a significant increase in surface chemical heterogeneity. Conclusions The surface chemical heterogeneity of the corneal epithelium may influence the dynamic behavior of tear film by “pinning” the contact line between the cellular surface and aqueous tear film. Engineering the surface properties of corneal epithelium could potentially lead to novel treatments in dry eye disease. PMID:26747762

  20. Ionizing radiation induces heritable disruption of epithelial cell interactions

    NASA Technical Reports Server (NTRS)

    Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Chatterjee, A. (Principal Investigator)

    2003-01-01

    Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

  1. Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells.

    PubMed

    Hegan, Peter S; Ostertag, Eric; Geurts, Aron M; Mooseker, Mark S

    2015-10-01

    In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost.

  2. Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

    PubMed Central

    Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. PMID:25861018

  3. Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

    PubMed Central

    2014-01-01

    Background Vinflunine (VFL) is a microtubule-targeting drug that suppresses microtubule dynamics, showing anti-metastatic properties both in vitro and in living cancer cells. An increasing body of evidence underlines the influence of the microtubules dynamics on the cadherin-dependent cell-cell adhesions. E-cadherin is a marker of epithelial-to-mesenchymal transition (EMT) and a tumour suppressor; its reduced levels in carcinoma are associated with poor prognosis. In this report, we investigate the role of VFL on cell-cell adhesions in bladder epithelial tumour cells. Methods Human bladder epithelial tumour cell lines HT1376, 5637, SW780, T24 and UMUC3 were used to analyse cadherin-dependent cell-cell adhesions under VFL treatment. VFL effect on growth inhibition was measured by using a MTT colorimetric cell viability assay. Western blot, immunofluorescence and transmission electron microscopy analyses were performed to assess the roles of VFL effect on cell-cell adhesions, epithelial-to-mesenchymal markers and apoptosis. The role of the proteasome in controlling cell-cell adhesion was studied using the proteasome inhibitor MG132. Results We show that VFL induces cell death in bladder cancer cells and activates epithelial differentiation of the remaining living cells, leading to an increase of E-cadherin-dependent cell-cell adhesion and a reduction of mesenchymal markers, such as N-cadherin or vimentin. Moreover, while E-cadherin is increased, the levels of Hakai, an E3 ubiquitin-ligase for E-cadherin, were significantly reduced in presence of VFL. In 5637, this reduction on Hakai expression was blocked by MG132 proteasome inhibitor, indicating that the proteasome pathway could be one of the molecular mechanisms involved in its degradation. Conclusions Our findings underscore a critical function for VFL in cell-cell adhesions of epithelial bladder tumour cells, suggesting a novel molecular mechanism by which VFL may impact upon EMT and metastasis. PMID:25012153

  4. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency.

    PubMed

    Utheim, Tor Paaske; Utheim, Øygunn Aass; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-01-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells. PMID:26938569

  5. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency

    PubMed Central

    Paaske Utheim, Tor; Aass Utheim, Øygunn; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-01-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells. PMID:26938569

  6. Sphere formation permits Oct4 reprogramming of ciliary body epithelial cells into induced pluripotent stem cells.

    PubMed

    Ni, Aiguo; Wu, Ming Jing; Chavala, Sai H

    2014-12-15

    Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by defined sets of transcription factors. We previously described reprogramming of monolayer-cultured adult mouse ciliary body epithelial (CE) cells by Oct4 and Klf4, but not with Oct4 alone. In this study, we report that Oct4 alone is sufficient to reprogram CE cells to iPS cells through sphere formation. Furthermore, we demonstrate that sphere formation induces a partial reprogramming state characterized by expression of retinal progenitor markers, upregulation of reprogramming transcription factors, such as Sall4 and Nanog, demethylation in the promoter regions of pluripotency associated genes, and mesenchymal to epithelial transition. The Oct4-iPS cells maintained normal karyotypes, expressed markers for pluripotent stem cells, and were capable of differentiating into derivatives of all three embryonic germ layers in vivo and in vitro. These findings suggest that sphere formation may render somatic cells more susceptible to reprogramming.

  7. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells.

    PubMed

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A; Costa, Max

    2016-02-15

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. PMID:26780400

  8. ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCTED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS

    EPA Science Inventory

    ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS.
    OBJECTIVE: We have shown that functional gap junction communication as measured by Lucifer yellow dye transfer (DT) in Clone-9 rat liver epithelial cells, c...

  9. Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

    PubMed Central

    Gibson, P; Birchall, I; Rosella, O; Albert, V; Finch, C; Barkla, D; Young, G

    1998-01-01

    Background—The functions of urokinase in intestinal epithelia are unknown. 
Aims—To determine the relation of urokinase expressed by intestinal epithelial cells to their position in the crypt-villus/surface axis and of mucosal urokinase activity to epithelial proliferative kinetics in the distal colon. 
Methods—Urokinase expression was examined immunohistochemically in human intestinal mucosa. Urokinase activity was measured colorimetrically in epithelial cells isolated sequentially from the crypt-villus axis of the rat small intestine. In separate experiments, urokinase activity and epithelial kinetics (measured stathmokinetically) were measured in homogenates of distal colonic mucosa of 14 groups of eight rats fed diets known to alter epithelial turnover. 
Results—From the crypt base, an ascending gradient of expression and activity of urokinase was associated with the epithelial cells. Median mucosal urokinase activities in each of the dietary groups of rats correlated positively with autologous median number of metaphase arrests per crypt (r=0.68; p<0.005) and per 100 crypt cells (r=0.75; p<0.001), but not with crypt column height. 
Conclusions—Localisation of an enzyme capable of leading to digestion of cell substratum in the region where cells are loosely attached to their basement membrane, and the association of its activity with indexes of cell turnover, suggest a role for urokinase in facilitating epithelial cell loss in the intestine. 

 Keywords: urokinase; intestinal epithelium; colon; epithelial proliferation PMID:9824347

  10. Roles of Wnt/{beta}-catenin signaling in epithelial differentiation of mesenchymal stem cells

    SciTech Connect

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

    2009-12-25

    Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/{beta}-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/{beta}-catenin signaling were determined, suggested down-regulation of Wnt/{beta}-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3{alpha} can inhibit the epithelial differentiation of MSCs. A loss of {beta}-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated {beta}-catenin expression and subsequently decreased {beta}-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/{beta}-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

  11. NADPH oxidase-dependent acid production in airway epithelial cells.

    PubMed

    Schwarzer, Christian; Machen, Terry E; Illek, Beate; Fischer, Horst

    2004-08-27

    The purpose of this study was to determine the role of NADPH oxidase in H(+) secretion by airway epithelia. In whole cell patch clamp recordings primary human tracheal epithelial cells (hTE) and the human serous gland cell line Calu-3 expressed a functionally similar zinc-blockable plasma membrane H(+) conductance. However, the rate of H(+) secretion of confluent epithelial monolayers measured in Ussing chambers was 9-fold larger in hTE compared with Calu-3. In hTE H(+) secretion was blocked by mucosal ZnCl(2) and the NADPH oxidase blockers acetovanillone and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), whereas these same blockers had no effect in Calu-3. We determined levels of transcripts for the NADPH oxidase transmembrane isoforms (Nox1 through -5, Duox1 and -2, and p22(phox)) and found Duox1, -2, and p22(phox) to be highly expressed in hTE, as well as the intracellular subunits p40(phox), p47(phox), and p67(phox). In contrast, Calu-3 lacked transcripts for Duox1, p40(phox), and p47(phox). Anti-Duox antibody staining resulted in prominent apical staining in hTE but no significant staining in Calu-3. When treated with amiloride to block the Na(+)/H(+) exchanger, intracellular pH in hTE acidified at significantly higher rates than in Calu-3, and treatment with AEBSF blocked acidification. These data suggest a role for an apically located Duox-based NADPH oxidase during intracellular H(+) production and H(+) secretion, but not in H(+) conduction.

  12. Spatiotemporally Regulated Ablation of Klf4 in Adult Mouse Corneal Epithelial Cells Results in Altered Epithelial Cell Identity and Disrupted Homeostasis

    PubMed Central

    Delp, Emili E.; Swamynathan, Sudha; Kao, Winston W.; Swamynathan, Shivalingappa K.

    2015-01-01

    Purpose. In previous studies, conditional disruption of Klf4 in the developing mouse ocular surface from embryonic day 10 resulted in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells, revealing the importance of Klf4 in ocular surface maturation. Here, we use spatiotemporally regulated ablation of Klf4 to investigate its functions in maintenance of adult corneal epithelial homeostasis. Methods. Expression of Cre was induced in ternary transgenic (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse corneal epithelium by doxycycline administered through intraperitoneal injections and drinking water, to generate corneal epithelium–specific deletion of Klf4 (Klf4Δ/ΔCE). Corneal epithelial barrier function was tested by fluorescein staining. Expression of selected Klf4-target genes was determined by quantitative PCR (QPCR), immunoblotting, and immunofluorescent staining. Results. Klf4 was efficiently ablated within 5 days of doxycycline administration in adult Klf4Δ/ΔCE corneal epithelium. The Klf4Δ/ΔCE corneal epithelial barrier function was disrupted, and the basal cells were swollen and rounded after 15 days of doxycycline treatment. Increased numbers of cell layers and Ki67-positive proliferating cells suggested deregulated Klf4Δ/ΔCE corneal epithelial homeostasis. Expression of tight junction proteins ZO-1 and occludin, desmosomal Dsg and Dsp, basement membrane laminin-332, and corneal epithelial–specific keratin-12 was decreased, while that of matrix metalloproteinase Mmp9 and noncorneal keratin-17 increased, suggesting altered Klf4Δ/ΔCE corneal epithelial cell identity. Conclusions. Ablation of Klf4 in the adult mouse corneas resulted in the absence of characteristic corneal epithelial cell differentiation, disrupted barrier function, and squamous metaplasia, revealing that Klf4 is essential for maintenance of the adult corneal epithelial cell identity and homeostasis. PMID:26047041

  13. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche.

    PubMed

    Davis, Hayley; Irshad, Shazia; Bansal, Mukesh; Rafferty, Hannah; Boitsova, Tatjana; Bardella, Chiara; Jaeger, Emma; Lewis, Annabelle; Freeman-Mills, Luke; Giner, Francesc C; Rodenas-Cuadrado, Pedro; Mallappa, Sreelakshmi; Clark, Susan; Thomas, Huw; Jeffery, Rosemary; Poulsom, Richard; Rodriguez-Justo, Manuel; Novelli, Marco; Chetty, Runjan; Silver, Andrew; Sansom, Owen J; Greten, Florian R; Wang, Lai Mun; East, James E; Tomlinson, Ian; Leedham, Simon J

    2015-01-01

    Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

  14. ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis

    PubMed Central

    Guo, Feiye; Ding, Ying; Caberoy, Nora; Alvarado, Gabriela; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis of shed photoreceptor outer segments (POSs) by retinal pigment epithelial (RPE) cells is critical to retinal homeostasis and shares many conserved signaling pathways with other phagocytes, including extrinsic regulations. Phagocytotic ligands are the key to cargo recognition, engulfment initiation, and activity regulation. In this study, we identified intracellular protein ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytotic ligand by a new approach of functional screening. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cells. This finding was further corroborated with primary RPE cells and RPE explants. Internalized POS vesicles were colocalized with a phagosome marker, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells and selectively bound to shed POS vesicles and apoptotic cells, possibly via externalized phosphatidylserine. ABCF1 is predominantly expressed in POSs and colocalized with the POS marker rhodopsin, providing geographical convenience for regulation of RPE phagocytosis. Collectively these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway. Furthermore, the new approach is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to understand extrinsic regulation and cargo recognition. PMID:25904329

  15. Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells.

    PubMed

    Rymut, Sharon M; Kampman, Claire M; Corey, Deborah A; Endres, Tori; Cotton, Calvin U; Kelley, Thomas J

    2016-08-01

    High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.

  16. Investigating the Responses of Human Epithelial Cells to Predatory Bacteria

    PubMed Central

    Monnappa, Ajay K.; Bari, Wasimul; Choi, Seong Yeol; Mitchell, Robert J.

    2016-01-01

    One beguiling alternative to antibiotics for treating multi-drug resistant infections are Bdellovibrio-and-like-organisms (BALOs), predatory bacteria known to attack human pathogens. Consequently, in this study, the responses from four cell lines (three human and one mouse) were characterized during an exposure to different predatory bacteria, Bdellovibrio bacteriovorus HD100, Bacteriovorus BY1 and Bacteriovorax stolpii EB1. TNF-α levels were induced in Raw 264.7 mouse macrophage cultures with each predator, but paled in comparison to those obtained with E. coli. This was true even though the latter strain was added at an 11.1-fold lower concentration (p < 0.01). Likewise, E. coli led to a significant (54%) loss in the Raw 264.7 murine macrophage viability while the predatory strains had no impact. Tests with various epithelial cells, including NuLi-1 airway, Caco2, HT29 and T84 colorectal cells, gave similar results, with E. coli inducing IL-8 production. The viabilities of the NuLi-1 and Caco-2 cells were slightly reduced (8%) when exposed to the predators, while T84 viability remained steady. In no cases did the predatory bacteria induce actin rearrangement. These results clearly demonstrate the gentle natures of predatory bacteria and their impacts on human cells. PMID:27629536

  17. Radiation-induced chromosomal instability in human mammary epithelial cells

    NASA Astrophysics Data System (ADS)

    Durante, M.; Grossi, G. F.; Yang, T. C.

    Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

  18. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    SciTech Connect

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  19. Investigating the Responses of Human Epithelial Cells to Predatory Bacteria.

    PubMed

    Monnappa, Ajay K; Bari, Wasimul; Choi, Seong Yeol; Mitchell, Robert J

    2016-01-01

    One beguiling alternative to antibiotics for treating multi-drug resistant infections are Bdellovibrio-and-like-organisms (BALOs), predatory bacteria known to attack human pathogens. Consequently, in this study, the responses from four cell lines (three human and one mouse) were characterized during an exposure to different predatory bacteria, Bdellovibrio bacteriovorus HD100, Bacteriovorus BY1 and Bacteriovorax stolpii EB1. TNF-α levels were induced in Raw 264.7 mouse macrophage cultures with each predator, but paled in comparison to those obtained with E. coli. This was true even though the latter strain was added at an 11.1-fold lower concentration (p < 0.01). Likewise, E. coli led to a significant (54%) loss in the Raw 264.7 murine macrophage viability while the predatory strains had no impact. Tests with various epithelial cells, including NuLi-1 airway, Caco2, HT29 and T84 colorectal cells, gave similar results, with E. coli inducing IL-8 production. The viabilities of the NuLi-1 and Caco-2 cells were slightly reduced (8%) when exposed to the predators, while T84 viability remained steady. In no cases did the predatory bacteria induce actin rearrangement. These results clearly demonstrate the gentle natures of predatory bacteria and their impacts on human cells. PMID:27629536

  20. ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis.

    PubMed

    Guo, Feiye; Ding, Ying; Caberoy, Nora; Alvarado, Gabriela; Wang, Feng; Chen, Rui; Li, Wei

    2015-06-15

    Phagocytosis of shed photoreceptor outer segments (POSs) by retinal pigment epithelial (RPE) cells is critical to retinal homeostasis and shares many conserved signaling pathways with other phagocytes, including extrinsic regulations. Phagocytotic ligands are the key to cargo recognition, engulfment initiation, and activity regulation. In this study, we identified intracellular protein ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytotic ligand by a new approach of functional screening. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cells. This finding was further corroborated with primary RPE cells and RPE explants. Internalized POS vesicles were colocalized with a phagosome marker, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells and selectively bound to shed POS vesicles and apoptotic cells, possibly via externalized phosphatidylserine. ABCF1 is predominantly expressed in POSs and colocalized with the POS marker rhodopsin, providing geographical convenience for regulation of RPE phagocytosis. Collectively these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway. Furthermore, the new approach is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to understand extrinsic regulation and cargo recognition.

  1. Radiation-induced chromosomal instability in human mammary epithelial cells

    NASA Technical Reports Server (NTRS)

    Durante, M.; Grossi, G. F.; Yang, T. C.

    1996-01-01

    Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

  2. Disruption of the keratin filament network during epithelial cell division.

    PubMed Central

    Lane, E B; Goodman, S L; Trejdosiewicz, L K

    1982-01-01

    The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6202508

  3. The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

    PubMed

    Valeri, Maria; Zurli, Vanessa; Ayala, Inmaculada; Colanzi, Antonino; Lapazio, Lucia; Corda, Daniela; Soriani, Marco; Pizza, Mariagrazia; Rossi Paccani, Silvia

    2015-01-01

    Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

  4. Prostate-regenerating capacity of cultured human adult prostate epithelial cells.

    PubMed

    Yao, M; Taylor, R A; Richards, M G; Sved, P; Wong, J; Eisinger, D; Xie, C; Salomon, R; Risbridger, G P; Dong, Q

    2010-01-01

    Experimentation with the progenitor/stem cells in adult prostate epithelium can be inconvenient due to a tight time line from tissue acquisition to cell isolation and to downstream experiments. To circumvent this inconvenience, we developed a simple technical procedure for culturing epithelial cells derived from human prostate tissue. In this study, benign prostate tissue was enzymatically digested and fractionated into epithelium and stroma, which were then cultured in the medium designed for prostate epithelial and stromal cells, respectively. The cultured cells were analyzed by immunocytochemical staining and flow cytometry. Prostate tissue-regenerating capacity of cultured cells in vitro was determined by co-culturing epithelial and stromal cells in dihydrotestosterone-containing RPMI. Cell lineages in formed acini-like structures were determined by immunohistochemistry. The culture of epithelial cells mainly consisted of basal cells. A minor population was negative for known lineage markers and positive for CD133. The culture also contained cells with high activity of aldehyde dehydrogenase. After co-culturing with stromal cells, the epithelial cells were able to form acini-like structures containing multiple cell lineages. Thus, the established culture of prostate epithelial cells provides an alternative source for studying progenitor/stem cells of prostate epithelium.

  5. Renal allograft rejection: investigation of alloantigen presentation by cultured human renal epithelial cells.

    PubMed Central

    Kirby, J A; Ikuta, S; Clark, K; Proud, G; Lennard, T W; Taylor, R M

    1991-01-01

    Defined lines of primary human renal epithelial cells were established and their expression of class II major histocompatibility (MHC) antigens was up-regulated by culture with interferon-gamma (IFN-gamma). The ability of these cells to stimulate the proliferation of allogeneic peripheral blood mononuclear cells (PBMC) was compared with that of endothelial cells and splenic mononuclear cells. It was found that both endothelial and splenic cells stimulated lympho-proliferation but that cultured renal epithelial cells were non-stimulatory. The failure of proliferation by allogeneic lymphocytes in culture with epithelial cells was not overcome by treatment with interleukin-1 (IL-1) or indomethacin. However, addition of IL-2 to mixed cultures of allogeneic PBMC and renal epithelial cells stimulated lympho-proliferation and allowed the generation of lymphoid cell lines which mediated non-specific lysis of renal epithelial cell lines. Stimulation of PBMC by mixed lymphocyte culture yielded an allospecific T-cell line which was added either to renal epithelial cells from the same donor as the stimulator cells used in the priming reaction or from a third-party donor; lympho-proliferation was observed in the specific secondary reaction but not in the non-specific reaction. These findings indicate that class II MHC antigen-expressing epithelial cells within a renal allograft may not initially stimulate the proliferation of resting allospecific recipient lymphocytes. However, within a rejecting graft it is likely that high local concentrations of IL-2 are present and that many of the infiltrating allospecific lymphocytes will be primed by previous contact with donor antigen-presenting cells, such as vascular endothelial cells or dendritic cells. Therefore, expression of class II MHC antigens by epithelial cells within the microenvironment of a renal allograft may render such cells immunogenic and able to play a direct role in the lymphocyte-mediated intragraft rejection

  6. Parietal Epithelial Cell Activation Marker in Early Recurrence of FSGS in the Transplant

    PubMed Central

    Fatima, Huma; Moeller, Marcus J.; Smeets, Bart; Yang, Hai-Chun; D’Agati, Vivette D.; Alpers, Charles E.

    2012-01-01

    Summary Background and objectives Podocyte loss is key in glomerulosclerosis. Activated parietal epithelial cells are proposed to contribute to pathogenesis of glomerulosclerosis and may serve as stem cells that can transition to podocytes. CD44 is a marker for activated parietal epithelial cells. This study investigated whether activated parietal epithelial cells are increased in early recurrent FSGS in transplant compared with minimal change disease. Design, setting, participants, & measurements CD44 staining in renal allograft biopsies from 12 patients with recurrent FSGS was performed and compared with native kidneys with minimal change disease or FSGS and normal control native and transplant kidneys without FSGS. CD44+ epithelial cells along Bowman’s capsule in the parietal epithelial cell location and over the glomerular tuft in the visceral epithelial cell location were assessed. Results Cases with early recurrent FSGS manifesting only foot process effacement showed significantly increased CD44+ visceral epithelial cells involving 29.0% versus 2.6% of glomeruli in minimal change disease and 0% in non-FSGS transplants. Parietal location CD44 positivity also was numerically increased in recurrent FSGS. In later transplant biopsies, glomeruli with segmental lesions had more CD44+ visceral epithelial cells than glomeruli without lesions. Conclusions Parietal epithelial cell activation marker is significantly increased in evolving FSGS versus minimal change disease, and this increase may distinguish early FSGS from minimal change disease. Whether parietal epithelial cell activation contributes to pathogenesis of sclerosis in idiopathic FSGS or is a regenerative/repair response to replace injured podocytes awaits additional study. PMID:22917699

  7. Intracellular killing of mastitis pathogens by penethamate hydriodide following internalization into mammary epithelial cells.

    PubMed

    Almeida, R A; Patel, D; Friton, G M; Oliver, S P

    2007-04-01

    Penethamate hydriodide was highly effective in killing Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Staphylococcus aureus that internalized mammary epithelial cells. At higher concentrations (32 microg/mL to 32 mg/mL), killing rates ranged from 85% to 100%. At lower concentrations (0.032 microg/mL to 3.2 microg/mL), killing rates ranged from 0 to 80%. Results of this proof-of-concept study demonstrated that: (1) penethamate hydriodide is capable of entering mammary epithelial cells and killing intracellular mastitis pathogens without affecting mammary epithelial cell viability, (2) the in vitro model used is capable of quantifying the fate of mastitis pathogens internalized into mammary epithelial cells, and (3) this in vitro model can be used to determine the effectiveness of antibiotics at killing bacteria within the cytoplasm of mammary epithelial cells.

  8. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

    PubMed

    Patil, Pratima U; D'Ambrosio, Julia; Inge, Landon J; Mason, Robert W; Rajasekaran, Ayyappan K

    2015-12-01

    In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells. PMID:26483386

  9. Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement.

    PubMed

    Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P; Cattin, Cedric J; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A; Hierlemann, Andreas; Müller, Daniel J

    2015-11-25

    Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.