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Sample records for epithelial cell cycle

  1. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression

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    Vielfort, Katarina; Weyler, Linda; Söderholm, Niklas; Engelbrecht, Mattias; Löfmark, Sonja; Aro, Helena

    2013-01-01

    We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells. PMID:23675492

  2. Lactobacillus decelerates cervical epithelial cell cycle progression.

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    Katarina Vielfort

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

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

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    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    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.

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

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

    1996-01-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation mediated by UV radiation. In these experiments, we investigated the effects of exposure to 254 nm radiation on cell cycle progression in the rabbit lens epithelial cell line N/N1003A. The RNA was harvested at various times following exposure to UV (254 nm) radiation and analyzed by dot-blot and northern blot hybridizations. These results revealed that during the first 6 h following exposure of the cells to UV, there was, associated with decreasing dose, a decrease in accumulation of transcripts specific for histones H3 and H4 and an increase in the mRNA encoding protein kinase C and β- and γ-actin. Using flow cytometry, we detected an accumulation of cells in G1/S phase of the cell cycle 1 h following exposure to 254 nm radiation. The observed changes in gene expression, especially the decreased accumulation of histone transcripts reported here, may play a role in UV-induced inhibition of cell cycle progression. (Author)

  5. Epithelial Label-Retaining Cells Are Absent during Tooth Cycling in Salmo salar and Polypterus senegalus.

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    Sam Vandenplas

    Full Text Available The Atlantic salmon (Salmo salar and African bichir (Polypterus senegalus are both actinopterygian fish species that continuously replace their teeth without the involvement of a successional dental lamina. Instead, they share the presence of a middle dental epithelium: an epithelial tier enclosed by inner and outer dental epithelium. It has been hypothesized that this tier could functionally substitute for a successional dental lamina and might be a potential niche to house epithelial stem cells involved in tooth cycling. Therefore, in this study we performed a BrdU pulse chase experiment on both species to (1 determine the localization and extent of proliferating cells in the dental epithelial layers, (2 describe cell dynamics and (3 investigate if label-retaining cells are present, suggestive for the putative presence of stem cells. Cells proliferate in the middle dental epithelium, outer dental epithelium and cervical loop at the lingual side of the dental organ to form a new tooth germ. Using long chase times, both in S. salar (eight weeks and P. senegalus (eight weeks and twelve weeks, we could not reveal the presence of label-retaining cells in the dental organ. Immunostaining of P. senegalus dental organs for the transcription factor Sox2, often used as a stem cell marker, labelled cells in the zone of outer dental epithelium which grades into the oral epithelium (ODE transition zone and the inner dental epithelium of a successor only. The location of Sox2 distribution does not provide evidence for epithelial stem cells in the dental organ and, more specifically, in the middle dental epithelium. Comparison of S. salar and P. senegalus reveals shared traits in tooth cycling and thus advances our understanding of the developmental mechanism that ensures lifelong replacement.

  6. Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

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    Chen, Jian; Guerriero, Emily; Lathrop, Kira; SundarRaj, Nirmala

    2008-01-01

    The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

  7. Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene

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    Gupta, Anu; Yang, L.-X.; Chen, L.-C.

    2002-01-01

    Purpose: Members of the cullin gene family are known to be involved in cell cycle control. One of the cullin genes, Cul-4A, is amplified and overexpressed in breast cancer cells. This study investigates the effect of Cul-4A overexpression upon G2/M cell cycle checkpoint after DNA damage induced by either ionizing or nonionizing radiation. Methods and Materials: The normal mammary epithelial cell line MCF10A was stably transfected with full-length Cul-4A cDNA. Independent clones of MCF10A cells that overexpress Cul-4A proteins were selected and treated with either 8 Gy of ionizing radiation or 7 J/M 2 of UV radiation. The profile of cell cycle progression and the accumulation of several cell cycle proteins were analyzed. Results: We found that overexpression of Cul-4A in MCF10A cells abrogated the G2/M cell cycle checkpoint in response to DNA damage induced by ionizing irradiation, but not to DNA damage induced by nonionizing radiation. Analysis of cell cycle proteins showed that after ionizing irradiation, p53 accumulated in the mock-transfected MCF10A cells, but not in the Cul-4A transfectants. Conclusion: Our results suggest a role for Cul-4A in tumorigenesis and/or tumor progression, possibly through disruption of cell cycle control

  8. CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

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    Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

    2006-05-01

    We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

  9. Cell cycle indicators of buccal epithelial cells in the treatment of different types of removable plate partial dentures

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    E. V. Beliaiev

    2018-02-01

    Full Text Available The purpose of the work. To investigate nuclear DNA and buccal epithelial cells proliferative activity in patients with dental defects, who use removable partial dentures plates made of acrylic or thermoplastic. Materials and Methods. The study of buccal epithelial cell cycle parameters was carried out in 70 people. Among them 23 patients were treated with acrylic dentures prostheses, 23 patients – with thermoplastic-based prostheses. The comparison group consisted of 24 clinically healthy persons without defects in the dentition. DNA content in human buccal epithelial cells nuclei was determined by flow cytometry. Results. The obtained indicators of buccal epithelial cell cycle of the control group indicate a high intensity of cell self-renewal in the normal range. It is suggested by a significant percentage of events occurring within the Sub-G1 range that characterizes apoptosis, as well as the fact that more than half of the cells were in the range of S + G2/M. It has been revealed by flow cytometry that the percentage of apoptosis in cells was higher in patients using acrylic dentures base plastic, showed initial signs of keratinization that was confirmed by increase in cells in the range of Sub-G1 and by their decrease in the range of S-G2/M. It has been established in the study of buccal epithelium cell cycle indicators in the dentures bases thermoplastic application that these prostheses did not affect the proliferative activity of buccal epithelial cells compared to the group using acrylic dentures bases with prolonged use. This is evident in almost the same number of cellular events ranging Sub-G1, so apoptosis in the thermoplastic dentures bases application corresponded to the control group indicators both in the early period and over a year of use. Conclusions. The direct negative effect of prostheses with acrylic bases on the complex mechanism of the oral cavity mucous membrane functioning has been revealed. Absence of dentures

  10. Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells

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    Morris Robert

    2010-03-01

    Full Text Available Abstract Background Sulforaphane (SFN, an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC. The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC. Results SFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose-polymerase (PARP. Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex. Conclusions SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.

  11. The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

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    Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

    2017-08-01

    Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

  12. The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

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    Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-01-01

    The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

  13. Flow cytometric analysis of mitotic cycle perturbation by chemical carcinogens in cultured epithelial cells. [Effects of benzo(a)pyrene-diol-epoxide on mitotic cycle of cultural mouse liver epithelial cells

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    Pearlman, Andrew Leonard [Univ. of California, Berkeley, CA (United States)

    1978-08-01

    A system for kinetic analysis of mitotic cycle perturbation by various agents was developed and applied to the study of the mitotic cycle effects and dependency of the chemical carcinogen benzo(a)pyrene-diolepoxide, DE, upon a mouse lever epithelial cell line, NMuLi. The study suggests that the targets of DE action are not confined to DNA alone but may include cytoplasmic structures as well. DE was found to affect cells located in virtually every phase of the mitotic cycle, with cells that were actively synthesizing DNA showing the strongest response. However, the resulting perturbations were not confined to S-phase alone. DE slowed traversal through S-phase by about 40% regardless of the cycle phase of the cells exposed to it, and slowed traversal through G2M by about 50%. When added to G1 cells, DE delayed recruitment of apparently quiescent (G0) cells by 2 hours, and reduced the synchrony of the cohort of cells recruited into active proliferation. The kinetic analysis system consists of four elements: tissue culture methods for propagating and harvesting cell populations; an elutriation centrifugation system for bulk synchronization of cells in various phases of the mitotic cycle; a flow cytometer (FCM), coupled with appropriate staining protocols, to enable rapid analysis of the DNA distribution of any given cell population; and data reduction and analysis methods for extracting information from the DNA histograms produced by the FCM. The elements of the system are discussed. A mathematical analysis of DNA histograms obtained by FCM is presented. The analysis leads to the detailed implementation of a new modeling approach. The new modeling approach is applied to the estimation of cell cycle kinetic parameters from time series of DNA histograms, and methods for the reduction and interpretation of such series are suggested.

  14. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

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    Biddle Adrian

    2010-04-01

    Full Text Available Abstract Background Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Methods Cells isolated from fresh human head and neck carcinomas (n = 11, cell lines derived from head and neck, prostate and breast human carcinomas (n = 7, and from normal human oral mucosa (n = 5, were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin. Flow cytometry for CD44 and epithelial-specific antigen (ESA expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR. The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH and siRNA. Results In both cancer biopsies and carcinoma cell lines a subset of CD44high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU demonstrated that CD44high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44high cells showing increased clonogenicity, and a similar pattern of G2-block

  15. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

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    Harper, Lisa J; Costea, Daniela Elena; Gammon, Luke; Fazil, Bilal; Biddle, Adrian; Mackenzie, Ian C

    2010-01-01

    Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Cells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA. In both cancer biopsies and carcinoma cell lines a subset of CD44 high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44 high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44 high cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance. These data

  16. Retinal Pigmented Epithelial Cells Obtained from Human Induced Pluripotent Stem Cells Possess Functional Visual Cycle Enzymes in Vitro and in Vivo*

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    Maeda, Tadao; Lee, Mee Jee; Palczewska, Grazyna; Marsili, Stefania; Tesar, Paul J.; Palczewski, Krzysztof; Takahashi, Masayo; Maeda, Akiko

    2013-01-01

    Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat−/− and Rpe65−/− mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat−/− and Rpe65−/− mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases. PMID:24129572

  17. Immunohistochemical analyses of cell cycle progression and gene expression of biliary epithelial cells during liver regeneration after partial hepatectomy of the mouse.

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    Fukuda, Tatsuya; Fukuchi, Tomokazu; Yagi, Shinomi; Shiojiri, Nobuyoshi

    2016-05-20

    The liver has a remarkable regeneration capacity, and, after surgical removal of its mass, the remaining tissue undergoes rapid regeneration through compensatory growth of its constituent cells. Although hepatocytes synchronously proliferate under the control of various signaling molecules from neighboring cells, there have been few detailed analyses on how biliary cells regenerate for their cell population after liver resection. The present study was undertaken to clarify how biliary cells regenerate after partial hepatectomy of mice through extensive analyses of their cell cycle progression and gene expression using immunohistochemical and RT-PCR techniques. When expression of PCNA, Ki67 antigen, topoisomerase IIα and phosphorylated histone H3, which are cell cycle markers, was immunohistochemically examined during liver regeneration, hepatocytes had a peak of the S phase and M phase at 48-72 h after resection. By contrast, biliary epithelial cells had much lower proliferative activity than that of hepatocytes, and their peak of the S phase was delayed. Mitotic figures were rarely detectable in biliary cells. RT-PCR analyses of gene expression of biliary markers such as Spp1 (osteopontin), Epcam and Hnf1b demonstrated that they were upregulated during liver regeneration. Periportal hepatocytes expressed some of biliary markers, including Spp1 mRNA and protein. Some periportal hepatocytes had downregulated expression of HNF4α and HNF1α. Gene expression of Notch signaling molecules responsible for cell fate decision of hepatoblasts to biliary cells during development was upregulated during liver regeneration. Notch signaling may be involved in biliary regeneration.

  18. Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells

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    Wang, Feng; Wang, Qi; Zhou, Zhi-Wei; Yu, Song-Ning; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Yin-Xue; Yang, Tianxing; Sun, Tao; Li, Min; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of

  19. Epithelial Cell Cultures

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    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  20. Characterisation of cell cycle arrest and terminal differentiation in a maximally proliferative human epithelial tissue: Lessons from the human hair follicle matrix.

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    Purba, Talveen S; Brunken, Lars; Peake, Michael; Shahmalak, Asim; Chaves, Asuncion; Poblet, Enrique; Ceballos, Laura; Gandarillas, Alberto; Paus, Ralf

    2017-09-01

    Human hair follicle (HF) growth and hair shaft formation require terminal differentiation-associated cell cycle arrest of highly proliferative matrix keratinocytes. However, the regulation of this complex event remains unknown. CIP/KIP family member proteins (p21 CIP1 , p27 KIP1 and p57 KIP2 ) regulate cell cycle progression/arrest, endoreplication, differentiation and apoptosis. Since they have not yet been adequately characterized in the human HF, we asked whether and where CIP/KIP proteins localise in the human hair matrix and pre-cortex in relation to cell cycle activity and HF-specific epithelial cell differentiation that is marked by keratin 85 (K85) protein expression. K85 expression coincided with loss or reduction in cell cycle activity markers, including in situ DNA synthesis (EdU incorporation), Ki-67, phospho-histone H3 and cyclins A and B1, affirming a post-mitotic state of pre-cortical HF keratinocytes. Expression of CIP/KIP proteins was found abundantly within the proliferative hair matrix, concomitant with a role in cell cycle checkpoint control. p21 CIP1 , p27 KIP1 and cyclin E persisted within post-mitotic keratinocytes of the pre-cortex, whereas p57 KIP2 protein decreased but became nuclear. These data imply a supportive role for CIP/KIP proteins in maintaining proliferative arrest, differentiation and anti-apoptotic pathways, promoting continuous hair bulb growth and hair shaft formation in anagen VI. Moreover, post-mitotic hair matrix regions contained cells with enlarged nuclei, and DNA in situ hybridisation showed cells that were >2N in the pre-cortex. This suggests that CIP/KIP proteins might counterbalance cyclin E to control further rounds of DNA replication in a cell population that has a propensity to become tetraploid. These data shed new light on the in situ-biography of human hair matrix keratinocytes on their path of active cell cycling, arrest and terminal differentiation, and showcase the human HF as an excellent, clinically

  1. Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

    Directory of Open Access Journals (Sweden)

    Qui JX

    2015-01-01

    critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT, and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for prostate cancer.Keywords: EMT, proteomics, SILAC

  2. Epithelial cell polarity, stem cells and cancer

    DEFF Research Database (Denmark)

    Martin-Belmonte, Fernando; Perez-Moreno, Mirna

    2011-01-01

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

  3. Cell volume regulation in epithelial physiology and cancer

    DEFF Research Database (Denmark)

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

    2013-01-01

    expression of ion transporters and channels is now recognized as one of the hallmarks of cancer, it is timely to consider this especially for epithelia. Epithelial cells are highly proliferative and epithelial cancers, carcinomas, account for about 90% of all cancers. In this review we will focus on ion...... such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered...... transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed....

  4. Andrographolide suppresses epithelial mesenchymal transition by inhibition of MAPK signalling pathway in lens epithelial cells.

    Science.gov (United States)

    Kayastha, Forum; Johar, Kaid; Gajjar, Devarshi; Arora, Anshul; Madhu, Hardik; Ganatra, Darshini; Vasavada, Abhay

    2015-06-01

    Epithelial mesenchymal transition (EMT) of lens epithelial cells (LECs) may contribute to the development of posterior capsular opacification (PCO), which leads to visual impairment. Andrographolide has been shown to have therapeutic potential against various cancers. However, its effect on human LECs is still unknown. The purpose of this study is to evaluate the effect of andrographolide on EMT induced by growth factors in the fetal human lens epithelial cell line (FHL 124). Initially the LECs were treated with growth factors (TGF-beta 2 and bFGF) to induce EMT. Subsequently these EMT-induced cells were treated with andrographolide at 100 and 500 nM concentrations for 24 h. Our results showed that FHL 124 cells treated with growth factors had a significant decrease in protein and m-RNA levels of epithelial markers pax6 and E-Cadherin. After administering andrographolide, these levels significantly increased. It was noticed that EMT markers alpha-SMA, fibronectin and collagen IV significantly decreased after treatment with andrographolide when compared to the other group. Treatment with andrographolide significantly inhibited phosphorylation of ERK and JNK. Cell cycle analysis showed that andrographolide did not arrest cells at G0/G1 or G2/M at tested concentrations. Our findings suggest that andrographolide helps sustain epithelial characteristics by modulating EMT markers and inhibiting the mitogen-activated protein kinase (MAPK) signalling pathway in LECs. Hence it can prove to be useful in curbing EMT-mediated PCO.

  5. Epithelial Cells in Urine: MedlinePlus Lab Test Information

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

  7. Patterning bacterial communities on epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Dwidar

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

  8. Human glomerular epithelial cell proteoglycans

    International Nuclear Information System (INIS)

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

    1990-01-01

    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

  9. Basolateral BMP signaling in polarized epithelial cells.

    Directory of Open Access Journals (Sweden)

    Masao Saitoh

    Full Text Available Bone morphogenetic proteins (BMPs regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER, counteracting reduction of TER by treatment with TGF-β or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.

  10. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  11. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

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

  12. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

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

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet....

  13. Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells.

    Science.gov (United States)

    Yoshioka, Masahiro; Ohashi, Shinya; Ida, Tomomi; Nakai, Yukie; Kikuchi, Osamu; Amanuma, Yusuke; Matsubara, Junichi; Yamada, Atsushi; Miyamoto, Shin'ichi; Natsuizaka, Mitsuteru; Nakagawa, Hiroshi; Chiba, Tsutomu; Seno, Hiroshi; Muto, Manabu

    2017-08-01

    Epidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells. Immortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated. Cells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR

  14. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

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

    1995-01-01

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

  15. Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells

    Science.gov (United States)

    Wang, Feng; Li, Hai; Yan, Xiao-Gang; Zhou, Zhi-Wei; Yi, Zhi-Gang; He, Zhi-Xu; Pan, Shu-Ting; Yang, Yin-Xue; Wang, Zuo-Zheng; Zhang, Xueji; Yang, Tianxing; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Pancreatic cancer is the most aggressive cancer worldwide with poor response to current therapeutics. Alisertib (ALS), a potent and selective Aurora kinase A inhibitor, exhibits potent anticancer effects in preclinical and clinical studies; however, the effect and underlying mechanism of ALS in the pancreatic cancer treatment remain elusive. This study aimed to examine the effects of ALS on cell growth, autophagy, and epithelial-to-mesenchymal transition (EMT) and to delineate the possible molecular mechanisms in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that ALS exerted potent cell growth inhibitory, pro-autophagic, and EMT-suppressing effects in PANC-1 and BxPC-3 cells. ALS remarkably arrested PANC-1 and BxPC-3 cells in G2/M phase via regulating the expression of cyclin-dependent kinases 1 and 2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. ALS concentration-dependently induced autophagy in PANC-1 and BxPC-3 cells, which may be attributed to the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinases 1 and 2 (Erk1/2) but activation of 5′-AMP-dependent kinase signaling pathways. ALS significantly inhibited EMT in PANC-1 and BxPC-3 cells with an increase in the expression of E-cadherin and a decrease in N-cadherin. In addition, ALS suppressed the expression of sirtuin 1 (Sirt1) and pre-B cell colony-enhancing factor/visfatin in both cell lines with a rise in the level of acetylated p53. These findings show that ALS induces cell cycle arrest and promotes autophagic cell death but inhibits EMT in pancreatic cancer cells with the involvement of PI3K/Akt/mTOR, p38 MAPK, Erk1/2, and Sirt1-mediated signaling pathways. Taken together, ALS may represent a promising anticancer drug for pancreatic cancer treatment. More studies are warranted to investigate other molecular targets and

  16. Heterogeneity of limbal basal epithelial progenitor cells.

    Science.gov (United States)

    Hayashida, Yasutaka; Li, Wei; Chen, Ying-Ting; He, Hua; Chen, Szu-yu; Kheirkah, Ahmad; Zhu, Ying-Tien; Matsumoto, Yukihiro; Tseng, Scheffer C G

    2010-11-01

    Although corneal epithelial stem cells (SCs) are located at the limbus between the cornea and the conjunctiva, not all limbal basal epithelial cells are SCs. Using 2 dispase digestions to remove different amounts of limbal basal epithelial cells for cross-sections, flat mounts, and cytospin preparations, double immunostaining to pancytokeratins (PCK) and vimentin (Vim) identified 3 p63+ epithelial progenitors such as PCK-/Vim+, PCK/Vim, and PCK-/Vim+ and 1 p63+ mesenchymal cell, PCK-/Vim+. PCK-/Vim- progenitors had the smallest cell size were 10-20 times more enriched on collagen I-coated dishes in the 5-minute rapid adherent fraction that contained the highest percentage of p63+ cells but the lowest percentage of cytokeratin12+ cells, and gave rise to high Ki67 labeling and vivid clonal growth. In contrast, PCK+/Vim+ and PCK+/Vim- progenitors were found more in the slow-adherent fraction and yielded poor clonal growth. PCK/Vim progenitors and clusters of PCK-/Vim+ mesenchymal cells, which were neither melanocytes nor Langerhans cells, were located in the limbal basal region. Therefore, differential expression of PCK and Vim helps identify small PCK-/Vim- cells as the most likely candidate for SCs among a hierarchy of heterogeneous limbal basal progenitors, and their close association with PCK-/Vim+ presumed "niche" cells.

  17. Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Glahn, Felix; Wiese, Jan; Foth, Heidi [Martin-Luther-University, Halle-Wittenberg, Institute of Environmental Toxicology, Halle/Saale (Germany); Schmidt-Heck, Wolfgang; Guthke, Reinhard [Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Jena (Germany); Zellmer, Sebastian; Gebhardt, Rolf [University of Leipzig, Institute of Biochemistry, Medical Faculty, Leipzig (Germany); Golka, Klaus; Degen, Gisela H.; Hermes, Matthias; Schormann, Wiebke; Brulport, Marc; Bauer, Alexander; Bedawy, Essam [IfADo, Leibniz Research Centre for Working Environment and Human Factors, Dortmund (Germany); Hergenroeder, Roland [ISAS, Institute for Analytical Sciences, Dortmund (Germany); Lehmann, Thomas [Translational Centre for Regenerative Medicine, Leipzig (Germany); Hengstler, Jan G. [IfADo, Leibniz Research Centre for Working Environment and Human Factors, Dortmund (Germany)

    2008-08-15

    Workers occupationally exposed to cadmium, cobalt and lead have been reported to have increased levels of DNA damage. To analyze whether in vivo relevant concentrations of heavy metals cause systematic alterations in RNA expression patterns, we performed a gene array study using primary normal human bronchial epithelial cells. Cells were incubated with 15{mu}g/l Cd(II), 25{mu}g/l Co(II) and 550{mu}g/l Pb(II) either with individual substances or in combination. Differentially expressed genes were filtered out and used to identify enriched GO categories as well as KEGG pathways and to identify transcription factors whose binding sites are enriched in a given set of promoters. Interestingly, combined exposure to Cd(II), Co(II) and Pb(II) caused a coordinated response of at least seven stress response-related transcription factors, namely Oct-1, HIC1, TGIF, CREB, ATF4, SRF and YY1. A stress response was further corroborated by up regulation of genes involved in glutathione metabolism. A second major response to heavy metal exposure was deregulation of the cell cycle as evidenced by down regulation of the transcription factors ELK-1 and the Ets transcription factor GABP, as well as deregulation of genes involved in purine and pyrimidine metabolism. A third and surprising response was up regulation of genes involved in steroid metabolism, whereby promoter analysis identified up regulation of SRY that is known to play a role in sex determination. A forth response was up regulation of xenobiotic metabolising enzymes, particularly of dihydrodiol dehydrogenases 1 and 2 (AKR1C1, AKR1C2). Incubations with individual heavy metals showed that the response of AKR1C1 and AKR1C2 was predominantly caused by lead. In conclusion, we have shown that in vivo relevant concentrations of Cd(II), Co(II) and Pb(II) cause a complex and coordinated response in normal human bronchial epithelial cells. This study gives an overview of the most responsive genes. (orig.)

  18. Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Wang F

    2015-01-01

    Full Text Available Feng Wang,1,2 Hai Li,3 Xiao-Gang Yan,4 Zhi-Wei Zhou,2 Zhi-Gang Yi,5 Zhi-Xu He,6 Shu-Ting Pan,7 Yin-Xue Yang,3 Zuo-Zheng Wang,1 Xueji Zhang,8 Tianxing Yang,9 Jia-Xuan Qiu,7 Shu-Feng Zhou21Department of Hepatobiliary Surgery, General Hospital, Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Colorectal Surgery, General Hospital, Ningxia Medical University, 4Department of Oncological Surgery, The First People’s Hospital of Yinchuan, 5Department of General Surgery, Changqing Yangehu Hospital, Yinchuan, 6Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 7Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 8Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 9Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USAAbstract: Pancreatic cancer is the most aggressive cancer worldwide with poor response to current therapeutics. Alisertib (ALS, a potent and selective Aurora kinase A inhibitor, exhibits potent anticancer effects in preclinical and clinical studies; however, the effect and underlying mechanism of ALS in the pancreatic cancer treatment remain elusive. This study aimed to examine the effects of ALS on cell growth, autophagy, and epithelial-to-mesenchymal transition (EMT and to delineate the possible molecular mechanisms in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that ALS exerted potent cell growth inhibitory, pro-autophagic, and EMT-suppressing effects in PANC-1 and BxPC-3 cells. ALS remarkably arrested PANC-1 and Bx

  19. Replication of cultured lung epithelial cells

    International Nuclear Information System (INIS)

    Guzowski, D.; Bienkowski, R.

    1986-01-01

    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 ( 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

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

    Science.gov (United States)

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

  1. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

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

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

  2. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    Directory of Open Access Journals (Sweden)

    Chi-Chin Sun

    Full Text Available 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. Uranium induces oxidative stress in lung epithelial cells

    International Nuclear Information System (INIS)

    Periyakaruppan, Adaikkappan; Kumar, Felix; Sarkar, Shubhashish; Sharma, Chidananda S.; Ramesh, Govindarajan T.

    2007-01-01

    Uranium compounds are widely used in the nuclear fuel cycle, antitank weapons, tank armor, and also as a pigment to color ceramics and glass. Effective management of waste uranium compounds is necessary to prevent exposure to avoid adverse health effects on the population. Health risks associated with uranium exposure includes kidney disease and respiratory disorders. In addition, several published results have shown uranium or depleted uranium causes DNA damage, mutagenicity, cancer and neurological defects. In the current study, uranium toxicity was evaluated in rat lung epithelial cells. The study shows uranium induces significant oxidative stress in rat lung epithelial cells followed by concomitant decrease in the antioxidant potential of the cells. Treatment with uranium to rat lung epithelial cells also decreased cell proliferation after 72 h in culture. The decrease in cell proliferation was attributed to loss of total glutathione and superoxide dismutase in the presence of uranium. Thus the results indicate the ineffectiveness of antioxidant system's response to the oxidative stress induced by uranium in the cells. (orig.)

  4. The human airway epithelial basal cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Neil R Hackett

    2011-05-01

    Full Text Available The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population.Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels.The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.

  5. Multiparameter Cell Cycle Analysis.

    Science.gov (United States)

    Jacobberger, James W; Sramkoski, R Michael; Stefan, Tammy; Woost, Philip G

    2018-01-01

    Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

  6. Transcriptomic response of goat mammary epithelial cells to Mycoplasma agalactiae challenge – a preliminary study

    DEFF Research Database (Denmark)

    Ogorevc, Jernej; Mihevc, Sonja Prpar; Hedegaard, Jakob

    2015-01-01

    Mycoplasma agalactiae (Ma) is one of the main aetiological agents of intramammary infections in small ruminants, causing contagious agalactia. To better understand the underlying disease patterns a primary goat mammary epithelial cell (pgMEC) culture was established from the mammary tissue......, steroid metabolism, fatty acid metabolism, apoptosis signalling, transcription regulation, and cell cycle regulation. Based on the results we suggest that mammary epithelial cells in vivo contribute to the immune system by the induced expression of cytokines and other chemotactic agents, activation...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-02-01

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

  8. Development of Thymic Epithelial Cells

    DEFF Research Database (Denmark)

    Ulyanchenko, Svetlana; Vaidya, Harsh J.; O'Neill, Kathy E.

    2016-01-01

    The thymus is the primary lymphoid organ in which the T cell repertoire is generated. The complex cellularity of this organ is uniquely designed to facilitate T cell development: defects in thymus development or function can cause immunodeficiencies ranging from the absence of T cell-mediated imm......The thymus is the primary lymphoid organ in which the T cell repertoire is generated. The complex cellularity of this organ is uniquely designed to facilitate T cell development: defects in thymus development or function can cause immunodeficiencies ranging from the absence of T cell......-mediated immunity to broad-spectrum autoimmune disease. Peak thymus size and output occurs early in life, after which the thymus undergoes a natural process of involution. This results in the progressive loss of functional thymus tissue and correspondingly in decreased production of new naïve T cells with age...... - contributing to the diminished capacity of the aged immune system to adequately respond to new antigenic challenge. Age-related thymic involutions, together with the thymic involutions associated with cytotoxic therapies (e.g., radio- or chemotherapy), have raised interest in development of clinically useful...

  9. Traction forces exerted by epithelial cell sheets

    International Nuclear Information System (INIS)

    Saez, A; Anon, E; Ghibaudo, M; Di Meglio, J-M; Hersen, P; Ladoux, B; Du Roure, O; Silberzan, P; Buguin, A

    2010-01-01

    Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

  10. Effects on g2/m phase cell cycle distribution and aneuploidy formation of exposure to a 60 Hz electromagnetic field in combination with ionizing radiation or hydrogen peroxide in l132 nontumorigenic human lung epithelial cells.

    Science.gov (United States)

    Jin, Hee; Yoon, Hye Eun; Lee, Jae-Seon; Kim, Jae-Kyung; Myung, Sung Ho; Lee, Yun-Sil

    2015-03-01

    The aim of the present study was to assess whether exposure to the combination of an extremely low frequency magnetic field (ELF-MF; 60 Hz, 1 mT or 2 mT) with a stress factor, such as ionizing radiation (IR) or H2O2, results in genomic instability in non-tumorigenic human lung epithelial L132 cells. To this end, the percentages of G2/M-arrested cells and aneuploid cells were examined. Exposure to 0.5 Gy IR or 0.05 mM H2O2 for 9 h resulted in the highest levels of aneuploidy; however, no cells were observed in the subG1 phase, which indicated the absence of apoptotic cell death. Exposure to an ELF-MF alone (1 mT or 2 mT) did not affect the percentages of G2/M-arrested cells, aneuploid cells, or the populations of cells in the subG1 phase. Moreover, when cells were exposed to a 1 mT or 2 mT ELF-MF in combination with IR (0.5 Gy) or H2O2 (0.05 mM), the ELF-MF did not further increase the percentages of G2/M-arrested cells or aneuploid cells. These results suggest that ELF-MFs alone do not induce either G2/M arrest or aneuploidy, even when administered in combination with different stressors.

  11. Transcriptional landscape of glomerular parietal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Sina A Gharib

    Full Text Available Very little is known about the function of glomerular parietal epithelial cells (PECs. In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire.

  12. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  13. Transcriptional profiling of putative human epithelial stem cells

    Directory of Open Access Journals (Sweden)

    Koçer Salih S

    2008-07-01

    Full Text Available Abstract Background Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells. Results Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells. Conclusion This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-14

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

  15. Nuclear microscopy of rat colon epithelial cells

    Science.gov (United States)

    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.

  16. Nuclear microscopy of rat colon epithelial cells

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

  17. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-10-15

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

  18. Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

    DEFF Research Database (Denmark)

    Bath, Chris; Yang, Sufang; Muttuvelu, Danson

    2013-01-01

    The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth......, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE......, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET)....

  19. Cellular Plasticity of Epithelial Cells-Cause of Metastasis

    National Research Council Canada - National Science Library

    Sukumar, Saraswati

    2005-01-01

    .... We present a novel concept that cancer epithelial cells, possibly of stem cell origin, have inherent cellular plasticity and can differentiate into endothelial cells and form microvessels that serve...

  20. Silk Film Topography Directs Collective Epithelial Cell Migration

    Science.gov (United States)

    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

  1. Turbulent Dynamics of Epithelial Cell Cultures

    Science.gov (United States)

    Blanch-Mercader, C.; Yashunsky, V.; Garcia, S.; Duclos, G.; Giomi, L.; Silberzan, P.

    2018-05-01

    We investigate the large length and long time scales collective flows and structural rearrangements within in vitro human bronchial epithelial cell (HBEC) cultures. Activity-driven collective flows result in ensembles of vortices randomly positioned in space. By analyzing a large population of vortices, we show that their area follows an exponential law with a constant mean value and their rotational frequency is size independent, both being characteristic features of the chaotic dynamics of active nematic suspensions. Indeed, we find that HBECs self-organize in nematic domains of several cell lengths. Nematic defects are found at the interface between domains with a total number that remains constant due to the dynamical balance of nucleation and annihilation events. The mean velocity fields in the vicinity of defects are well described by a hydrodynamic theory of extensile active nematics.

  2. Molecular Characterization of Gastric Epithelial Cells Using Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Kevin A. Bockerstett

    2018-04-01

    Full Text Available The ability to analyze individual epithelial cells in the gastric mucosa would provide important insight into gastric disease, including chronic gastritis and progression to gastric cancer. However, the successful isolation of viable gastric epithelial cells (parietal cells, neck cells, chief cells, and foveolar cells from gastric glands has been limited due to difficulties in tissue processing. Furthermore, analysis and interpretation of gastric epithelial cell flow cytometry data has been difficult due to the varying sizes and light scatter properties of the different epithelial cells, high levels of autofluorescence, and poor cell viability. These studies were designed to develop a reliable method for isolating viable single cells from the corpus of stomachs and to optimize analyses examining epithelial cells from healthy and diseased stomach tissue by flow cytometry. We performed a two stage enzymatic digestion in which collagenase released individual gastric glands from the stromal tissue of the corpus, followed by a Dispase II digestion that dispersed these glands into greater than 1 × 106 viable single cells per gastric corpus. Single cell suspensions were comprised of all major cell lineages found in the normal gastric glands. A method describing light scatter, size exclusion, doublet discrimination, viability staining, and fluorescently-conjugated antibodies and lectins was used to analyze individual epithelial cells and immune cells. This technique was capable of identifying parietal cells and revealed that gastric epithelial cells in the chronically inflamed mucosa significantly upregulated major histocompatibility complexes (MHC I and II but not CD80 or CD86, which are costimulatory molecules involved in T cell activation. These studies describe a method for isolating viable single cells and a detailed description of flow cytometric analysis of cells from healthy and diseased stomachs. These studies begin to identify effects of

  3. induced acute cytotoxicity in human cervical epithelial carcinoma cells

    African Journals Online (AJOL)

    Molecular basis of arsenite (As +3 )-induced acute cytotoxicity in human cervical epithelial carcinoma cells. ... Libyan Journal of Medicine ... Methods: After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and ...

  4. Globoside accelerates the differentiation of dental epithelial cells into ameloblasts

    Institute of Scientific and Technical Information of China (English)

    Takashi Nakamura; Yuta Chiba; Masahiro Naruse; Kan Saito; Hidemitsu Harada; Satoshi Fukumoto

    2016-01-01

    Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

  5. Fibrosis of Two: Epithelial Cell-Fibroblast Interactions in Pulmonary Fibrosis

    Science.gov (United States)

    Sakai, Norihiko; Tager, Andrew M.

    2013-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E2, angiotensin II and reactive oxygen species. PMID:23499992

  6. Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model.

    Science.gov (United States)

    Gao, Yang; Li, Shu; Li, Qinglei

    2014-08-01

    In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Cytomatrix synthesis in MDCK epithelial cells

    International Nuclear Information System (INIS)

    Mitchell, J.J.; Low, R.B.; Woodcock-Mitchell, J.L.

    1990-01-01

    Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak, was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with [14C]leucine over several days and then pulse-labeled for 4 hours with [3H]leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form

  8. Canine tracheal epithelial cells are more sensitive than rat tracheal epithelial cells to transforming growth factor beta induced growth inhibition

    International Nuclear Information System (INIS)

    Hubbs, A.F.; Hahn, F.F.; Kelly, G.; Thomassen, D.G.

    1988-01-01

    Transforming growth factor beta (TGFβ) markedly inhibited growth of canine tracheal epithelial (CTE) cells. Reduced responsiveness to TGFβ-induced growth inhibition accompanied neoplastic progression of these cells from primary to transformed to neoplastic. This was similar to the relationship between neoplastic progression and increased resistance to TGFβ-induced growth inhibition seen for rat tracheal epithelial (RTE) cells. The canine cells were more sensitive than rat cells to TGFβ-induced growth inhibition at all stages in the neoplastic process. (author)

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

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

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

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

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

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

  11. No junctional communication between epithelial cells in hydra

    DEFF Research Database (Denmark)

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

    1980-01-01

    junctions between epithelial cells of hydra. However, until now, there has been no report published on whether these junctions enable the epithelial cells to exchange molecules of small molecular weight, as has been described in other organisms. Therefore we decided to investigate the communicative...... properties of the junctional membranes by electrophysiological methods and by intracellular-dye iontophoresis. We report here that no electrotonic coupling is detectable between epithelial cells of Hydra attenuata in: (1) intact animals, (2) head-regenerating animals, (3) cell re-aggregates, and (4) hydra...

  12. Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail.

    Science.gov (United States)

    Wei, Zhengxi; Shan, Zhongguo; Shaikh, Zahir A

    2018-04-01

    Epidemiological and experimental studies have implicated cadmium (Cd) with breast cancer. In breast epithelial MCF10A and MDA-MB-231 cells, Cd has been shown to promote cell growth. The present study examined whether Cd also promotes epithelial-mesenchymal transition (EMT), a hallmark of cancer progression. Human breast epithelial cells consisting of non-cancerous MCF10A, non-metastatic HCC 1937 and HCC 38, and metastatic MDA-MB-231 were treated with 1 or 3 μM Cd for 4 weeks. The MCF10A epithelial cells switched to a more mesenchymal-like morphology, which was accompanied by a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal markers N-cadherin and vimentin. In both non-metastatic HCC 1937 and HCC 38 cells, treatment with Cd decreased the epithelial marker claudin-1. In addition, E-cadherin also decreased in the HCC 1937 cells. Even the mesenchymal-like MDA-MB-231 cells exhibited an increase in the mesenchymal marker vimentin. These changes indicated that prolonged treatment with Cd resulted in EMT in both normal and cancer-derived breast epithelial cells. Furthermore, both the MCF10A and MDA-MB-231 cells labeled with Zcad, a dual sensor for tracking EMT, demonstrated a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker ZEB-1. Treatment of cells with Cd significantly increased the level of Snail, a transcription factor involved in the regulation of EMT. However, the Cd-induced Snail expression was completely abolished by actinomycin D. Luciferase reporter assay indicated that the expression of Snail was regulated by Cd at the promotor level. Snail was essential for Cd-induced promotion of EMT in the MDA-MB-231 cells, as knockdown of Snail expression blocked Cd-induced cell migration. Together, these results indicate that Cd promotes EMT in breast epithelial cells and does so by modulating the transcription of Snail. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Araki Hiromasa

    2007-04-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  15. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Raquel Rodrigues-Diez

    2014-01-01

    Full Text Available Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β. Epithelial mesenchymal transition (EMT is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2 with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription. The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β.

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Sodium selectivity of Reissner's membrane epithelial cells

    Directory of Open Access Journals (Sweden)

    Kim Kyunghee X

    2011-02-01

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

  18. Regulated Mucin Secretion from Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Kenneth Bruce Adler

    2013-09-01

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

  19. Epithelial cell proliferation arrest induced by lactate and acetate from Lactobacillus casei and Bifidobacterium breve.

    Directory of Open Access Journals (Sweden)

    Takahiro Matsuki

    Full Text Available In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either Lactobacillus or Bifidobacterium as bona fide symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by L. casei and B. breve respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.

  20. Role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers.

    Science.gov (United States)

    Selman, Moisés; Pardo, Annie

    2006-06-01

    Idiopathic pulmonary fibrosis (IPF), a progressive and relentless lung scarring of unknown etiology, has been recognized as the most lethal interstitial lung disease. Despite the growing interest in IPF, the precise molecular mechanisms underlying the development of fibrosis and leading to the irreversible destruction of the lung are still unknown. Recently, it has been proposed that IPF, instead of being a chronic inflammatory disorder, results from multiple cycles of epithelial cell injury and activation. In turn, active alveolar epithelial cells provoke the migration, proliferation, and activation of mesenchymal cells with the formation of fibroblastic/myofibroblastic foci and the exaggerated accumulation of extracellular matrix, mirroring abnormal wound repair. In this article, some characteristics of the alveolar epithelium are briefly outlined, and the fibrogenic mechanisms specifically operated by active abnormal epithelial cells are examined.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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 TGFβ1-mediated lytic phase. EBV lytic reactivation by TGFβ1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-07-01

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

  3. Dexmedetomidine Attenuates Oxidative Stress Induced Lung Alveolar Epithelial Cell Apoptosis In Vitro

    Directory of Open Access Journals (Sweden)

    Jian Cui

    2015-01-01

    Full Text Available Background. Oxidative stress plays a pivotal role in the lung injuries of critical ill patients. This study investigates the protection conferred by α2 adrenoceptor agonist dexmedetomidine (Dex from lung alveolar epithelial cell injury induced by hydrogen peroxide (H2O2 and the underlying mechanisms. Methods. The lung alveolar epithelial cell line, A549, was cultured and then treated with 500 μM H2O2 with or without Dex (1 nM or Dex in combination with atipamezole (10 nM, an antagonist of α2 receptors. Their effect on mitochondrial membrane potential (Δψm, reactive oxygen species (ROS, and the cell cycle was assessed by flow cytometry. Cleaved-caspases 3 and 9, BAX, Bcl-2, phospho-mTOR (p-mTOR, ERK1/2, and E-cadherin expression were also determined with immunocytochemistry. Results. Upregulation of cleaved-caspases 3 and 9 and BAX and downregulation of Bcl-2, p-mTOR, and E-cadherin were found following H2O2 treatment, and all of these were reversed by Dex. Dex also prevented the ROS generation, cytochrome C release, and cell cycle arrest induced by H2O2. The effects of Dex were partially reversed by atipamezole. Conclusion. Our study demonstrated that Dex protected lung alveolar epithelial cells from apoptotic injury, cell cycle arrest, and loss of cell adhesion induced by H2O2 through enhancing the cell survival and proliferation.

  4. Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

    OpenAIRE

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

    2011-01-01

    Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell proper...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-10-13

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

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

    International Nuclear Information System (INIS)

    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 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-β 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 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  7. Cell Cycle Control by PTEN.

    Science.gov (United States)

    Brandmaier, Andrew; Hou, Sheng-Qi; Shen, Wen H

    2017-07-21

    Continuous and error-free chromosome inheritance through the cell cycle is essential for genomic stability and tumor suppression. However, accumulation of aberrant genetic materials often causes the cell cycle to go awry, leading to malignant transformation. In response to genotoxic stress, cells employ diverse adaptive mechanisms to halt or exit the cell cycle temporarily or permanently. The intrinsic machinery of cycling, resting, and exiting shapes the cellular response to extrinsic stimuli, whereas prevalent disruption of the cell cycle machinery in tumor cells often confers resistance to anticancer therapy. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and a guardian of the genome that is frequently mutated or deleted in human cancer. Moreover, it is increasingly evident that PTEN deficiency disrupts the fundamental processes of genetic transmission. Cells lacking PTEN exhibit cell cycle deregulation and cell fate reprogramming. Here, we review the role of PTEN in regulating the key processes in and out of cell cycle to optimize genomic integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin

    International Nuclear Information System (INIS)

    Norgaard, J.O.

    1987-01-01

    Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of [ 3 H]thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of [ 3 H]thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with [ 3 H]thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium

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

    Science.gov (United States)

    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.

  10. Role of dihydrotestosterone (DHT) on TGF-β1 signaling pathway in epithelial ovarian cancer cells.

    Science.gov (United States)

    Kohan-Ivani, Karla; Gabler, Fernando; Selman, Alberto; Vega, Margarita; Romero, Carmen

    2016-01-01

    One of the hypotheses regarding the genesis of epithelial ovarian cancer involves the action of androgens on the proliferation of epithelial ovarian cells, as well as inclusion cysts. The purpose of the present study was to evaluate whether DHT causes changes in the TGF-β1 pathway that might modify the anti-proliferative effect of the latter. The levels of TGF-β1 protein, of its receptors (TGFBR1 and TGFBR2), of Smad2/3 (canonical signaling pathway protein) and of p21 (cell cycle protein) were assessed in ovarian tissues, epithelial ovarian cancer cell lines (A2780) and control cell lines (HOSE) through the use of immunohistochemistry and immunocytochemistry. Additionally, cell lines were treated with 100 nmol/L DHT, 10 ng/mL of TGF-β1 and DHT + TGF-β1 during 72 h in the presence and absence of a siRNA against androgen receptor. After treatment, TGFBR1 and TGFBR2 levels were detected through Western blotting and p21 was assessed through immunocytochemistry. Epithelial ovarian cancer tissues showed a decrease in TGF-β1 I receptor (p DHT, protein levels of TGF-β1 receptors (TGFBR1-TGFBR2) showed a decrease (p DHT (p < 0.001). Overall, our results indicate a defect in the canonical TGF-β signaling pathway in epithelial ovarian cancer caused by androgen action, thus suggesting eventual changes in such tissue proliferation rates.

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

    Science.gov (United States)

    Ishihara, Shuji; Marcq, Philippe; Sugimura, Kaoru

    2017-08-01

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

  12. Human Bronchial Epithelial Cell Response to Heavy Particle Exposure

    Science.gov (United States)

    Story, Michael; Ding, Liang-Hao; Minna, John; Park, Seong-mi; Peyton, Michael; Larsen, Jill

    2012-07-01

    A battery of non-oncogenically immortalized human bronchial epithelial cells (HBECs) are being used to examine the molecular changes that lead to lung carcinogenesis after exposure to heavy particles found in the free space environment. The goal is to ultimately identify biomarkers of radioresponse that can be used for prediction of carcinogenic risk for fatal lung cancer. Our initial studies have focused on the cell line HBEC3 KT and the isogenic variant HBEC3 KTR53, which overexpresses the RASv12 mutant and where p53 has been knocked down by shRNA, and is considered to be a more oncogenically progressed variant. We have previously described the response of HBEC3 KT at the cellular and molecular level, however, the focus here is on the rate of cellular transformation after HZE radiation exposure and the molecular changes in transformed cells. When comparing the two cell lines we find that there is a maximum rate of cellular transformation at 0.25 Gy when cells are exposed to 1 GeV Fe particles, and, for the HBEC3 KTR53 there are multiple pathways upregulated that promote anchorage independent growth including the mTOR pathway, the TGF-1 pathway, RhoA signaling and the ERK/MAPK pathway as early as 2 weeks after radiation. This does not occur in the HBEC3 KT cell line. Transformed HBEC3 KT cells do not show any morphologic or phenotypic changes when grown as cell cultures. HBEC3 KTR53 cells on the other hand show substantial changes in morphology from a cobblestone epithelial appearance to a mesenchymal appearance with a lack of contact inhibition. This epithelial to mesenchymal change in morphology is accompanied by the expression of vimentin and a reduction in the expression of E-cadherin, which are hallmarks of epithelial to mesenchymal transition. Interestingly, for HBEC3 KT transformed cells there are no mutations in the p53 gene, 2 of 15 clones were found to be heterozygous for the RASV12 mutation, and 3 of 15 clones expressed high levels of BigH3, a TGFB

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-10-15

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  15. Probiotics promote endocytic allergen degradation in gut epithelial cells

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  16. Probiotics promote endocytic allergen degradation in gut epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-14

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

  17. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  18. The regenerative potential of parietal epithelial cells in adult mice

    NARCIS (Netherlands)

    Berger, K.; Schulte, K.; Boor, P.; Kuppe, C.; Kuppevelt, T.H. van; Floege, J.; Smeets, B.; Moeller, M.J.

    2014-01-01

    Previously, we showed that some podocytes in juvenile mice are recruited from cells lining Bowman's capsule, suggesting that parietal epithelial cells (PECs) are a progenitor cell population for podocytes. To investigate whether PECs also replenish podocytes in adult mice, PECs were genetically

  19. Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis

    DEFF Research Database (Denmark)

    Wendland, Kerstin; Niss, Kristoffer; Kotarsky, Knut

    2018-01-01

    Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the abse......Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis...

  20. The kinetics of epithelial cell generation: its relevance to cancer and ageing.

    Science.gov (United States)

    Morris, J A

    1999-07-07

    A hierarchical model of epithelial cell generation is proposed, in which even in extreme old age mature epithelial cells in humans are only a limited number of cell divisions from the zygote (60-120). This contrasts with conventional models in which regularly cycling stem cells can be several thousands of cell divisions from the zygote. The hierarchical model is supported by data on the rate of telomere shortening both in vivo and in vitro, and by data on the rate of synonymous substitutions in Y-linked, X-linked and autosomal genes in rodents. Limiting the number of cell generations leads to a vast reduction in the risk of cancer and reduces the rate of ageing. It is suggested that longer-lived animals need stricter control of the hierarchy than do shorter-lived animals and this difference has implications for theories of ageing. Copyright 1999 Academic Press.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-06-26

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

  2. Cell growth and division cycle

    International Nuclear Information System (INIS)

    Darzynkiewicz, Z.

    1986-01-01

    The concept of the cell cycle in its present form was introduced more than three decades ago. Studying incorporation of DNA precursors by autoradiography, these authors observed that DNA synthesis in individual cells was discontinuous and occupied a discrete portion of the cell life (S phase). Mitotic division was seen to occur after a certain period of time following DNA replication. A distinct time interval between mitosis and DNA replication was also apparent. Thus, the cell cycle was subdivided into four consecutive phases, G/sub 1/, S, G/sub 2/, and M. The G/sub 1/ and G/sub 2/ phases represented the ''gaps'' between mitosis and the start of DNA replication, and between the end of DNA replication and the onset of mitosis, respectively. The cell cycle was defined as the interval between the midpoint of mitosis and the midpoint of the subsequent mitosis of the daughter cell(s). The authors' present knowledge on the cell cycle benefited mostly from the development of four different techniques: autoradiography, time-lapse cinematography, cell synchronization and flow cytometry. Of these, autoradiography has been the most extensively used, especially during the past two decades. By providing a means to analyse incorporation of precursors of DNA, RNA or proteins by individual cells and, in combination with various techniques of cell synchronization, autoradiography yielded most of the data fundamental to the current understanding of the cell cycle-related phenomena. Kinetics of cell progression through the cell cycle could be analysed in great detail after development of such sophisticated autoradiographic approaches as measurements of the fraction of labeled mitoses (''FLM curves'') or multiple sequential cell labelling with /sup 3/H- and /sup 14/C-TdR

  3. Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ya C Wu

    Full Text Available Hydrogen sulfide (H(2S is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC and a panel of colon cancer cell lines (HT-29, SW1116, HCT116 were exposed to H(2S at concentrations similar to those found in the human colon. H(2S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2S was accompanied by G(1-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip. Moreover, exposure to H(2S led to features characteristic of autophagy, including increased formation of LC3B(+ autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2S. Further mechanistic investigation revealed that H(2S stimulated the phosphorylation of AMP-activated protein kinase (AMPK and inhibited the phosphorylation of mammalian target of rapamycin (mTOR and S6 kinase. Inhibition of AMPK significantly reversed H(2S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

  4. Cell-cycle regulatory proteins in human wound healing

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik

    2003-01-01

    Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell......-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1......) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas...

  5. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells

    Directory of Open Access Journals (Sweden)

    Yuan CX

    2015-03-01

    autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K/protein kinase B (Akt/mammalian target of rapamycin (mTOR and p38 mitogen-activated protein kinase pathways as well as activation of 5' AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression of N-cadherin in both cell lines. Taken together, danusertib has potent inducing effects on cell cycle arrest, apoptosis, and autophagy, but has an inhibitory effect on epithelial to mesenchymal transition, with involvement of signaling pathways mediated by PI3K/Akt/mTOR, p38 mitogen-activated protein kinase, and 5' AMP-activated protein kinase in AGS and NCI-N78 cells. Keywords: danusertib, gastric cancer, Aurora kinase, apoptosis, autophagy, epithelial to mesenchymal transition

  6. Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis.

    LENUS (Irish Health Repository)

    O'Connell, J

    2012-02-03

    Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell\\'s sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95\\/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.

  7. Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Liara M Gonzalez

    Full Text Available Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA, Minichromosome Maintenance Complex 2 (MCM2, Bromodeoxyuridine (BrdU and phosphorylated Histone H3 (pH3 distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2; enteroendocrine cells by Chromogranin A (CGA, Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII and sucrase isomaltase (SIM. Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.

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

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

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

  9. Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

    Science.gov (United States)

    Cousins, Fiona L; Murray, Alison; Esnal, Arantza; Gibson, Douglas A; Critchley, Hilary O D; Saunders, Philippa T K

    2014-01-01

    In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These

  10. ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells.

    Science.gov (United States)

    Yan, Hongchao; Qiu, Linglin; Xie, Xiaolei; Yang, He; Liu, Yongli; Lin, Xiaoman; Huang, Hongxiang

    2017-03-01

    The expression of E74-like factor 5 (ELF5) in epithelial ovarian carcinoma tissues and its effects on biological behavior in ovarian carcinoma cells were assessed in search for a new approach for gene treatment of epithelial ovarian carcinoma. RT-PCR technology was applied to detect the expression of ELF5 mRNA in epithelial ovarian carcinoma (n=49), borderline ovarian epithelial tumor (n=19), benign ovarian epithelial tumor (n=31) and normal ovarian tissues (n=40). Then, we transfected recombinant plasmid pcDNA3.1‑ELF5+EGFP into human ovarian carcinoma SKOV3 cells (recombinant plasmid group) in vitro and screened out stably transfected cells to conduct multiplication culture. Western blot analysis was performed to detect the expression of ELF5 protein in the different groups. Flow cytometry was employed to detect cell apoptosis and cycles. ELF5 mRNA in epithelial ovarian carcinoma and borderline ovarian epithelial tumor tissues were significantly lower (Pepithelial tumor and normal ovarian tissues. ELF5 protein expression in the cells of recombinant plasmid group was significantly higher compared with empty plasmid and blank control groups. The capacity of cell reproductive recombinant plasmid group at each time point decreased (P<0.05). Flow cytometry detection showed that 67.03% of cells in recombinant plasmid group was blocked in G0/G1 phase (P<0.05), compared with empty plasmid group (37.17%) and blank control group (38.24%). Apoptotic rate of recombinant plasmid group was significantly lower (31.4±1.9%; P<0.05), compared with that of empty plasmid group (9.1±2.2%) and blank control group (8.7±1.5%), and the differences were statistically significant. In conclusion, ELF5 interfered with cell cycle of human ovarian carcinoma SKOV3 cells and promoted apoptosis of human ovarian carcinoma SKOV3 cells inhibiting their growth and invasive capacity; and thus providing a new approach to gene treatment of ovarian carcinoma.

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

    Indian Academy of Sciences (India)

    In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the ...

  12. Sphingolipid trafficking and protein sorting in epithelial cells

    NARCIS (Netherlands)

    Slimane, TA; Hoekstra, D

    2002-01-01

    Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an

  13. Studies in human skin epithelial cell carcinogenesis

    International Nuclear Information System (INIS)

    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 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 32 P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts

  14. Role of Bone Morphogenetic Protein 7 (BMP7 in the Modulation of Corneal Stromal and Epithelial Cell Functions

    Directory of Open Access Journals (Sweden)

    Bhavani S. Kowtharapu

    2018-05-01

    Full Text Available In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we

  15. Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells.

    NARCIS (Netherlands)

    Dijkman, H.B.P.M.; Weening, J.J.; Smeets, B.; Verrijp, K.; Kuppevelt, A.H.M.S.M. van; Assmann, K.K.; Steenbergen, E.; Wetzels, J.F.M.

    2006-01-01

    Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by hyperplasia of glomerular epithelial cells. In a mouse model of FSGS and in a patient with recurrent idiopathic FSGS, we identified the proliferating cells as parietal epithelial cells (PECs). In the present study, we have

  16. Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells

    NARCIS (Netherlands)

    Dijkman, H. B. P. M.; Weening, J. J.; Smeets, B.; Verrijp, K. C. N.; van Kuppevelt, T. H.; Assmann, K. K. J. M.; Steenbergen, E. J.; Wetzels, J. F. M.

    2006-01-01

    Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by hyperplasia of glomerular epithelial cells. In a mouse model of FSGS and in a patient with recurrent idiopathic FSGS, we identified the proliferating cells as parietal epithelial cells (PECs). In the present study, we have

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

    Science.gov (United States)

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

    2017-11-01

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

  18. Oral epithelial cell reaction after exposure to Invisalign plastic material.

    Science.gov (United States)

    Premaraj, Thyagaseely; Simet, Samantha; Beatty, Mark; Premaraj, Sundaralingam

    2014-01-01

    Invisalign plastic aligners (Align Technology, Santa Clara, Calif) are used to correct malocclusions. The aligners wrap around the teeth and are in contact with gingival epithelium during treatment. The purpose of this study was to evaluate the cellular responses of oral epithelium exposed to Invisalign plastic in vitro. Oral epithelial cells were exposed to eluate obtained by soaking Invisalign plastic in either saline solution or artificial saliva for 2, 4, and 8 weeks. Cells grown in media containing saline solution or saliva served as controls. Morphologic changes were assessed by light microscopy. The 3-[4, 5-dimethythiazol- 2-yl]-2, 5-diphenyl tetrazolium bromide assay and flow cytometry were used to determine cell viability and membrane integrity, respectively. Cellular adhesion and micromotion of epithelial cells were measured in real time by electrical cell-substrate impedance sensing. Cells exposed to saline-solution eluate appeared rounded, were lifted from the culture plates, and demonstrated significantly increased metabolic inactivity or cell death (P <0.05). Saliva eluates did not induce significant changes in cell viability compared with untreated cells. Flow cytometry and electric cell-substrate impedance sensing showed that cells treated with saline-solution eluate exhibited compromised membrane integrity, and reduced cell-to-cell contact and mobility when compared with saliva-eluate treatment. Exposure to Invisalign plastic caused changes in viability, membrane permeability, and adhesion of epithelial cells in a saline-solution environment. Microleakage and hapten formation secondary to compromised epithelial integrity might lead to isocyanate allergy, which could be systemic or localized to gingiva. However, these results suggest that saliva might offer protection. Copyright © 2014 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

  19. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

    Science.gov (United States)

    Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

    2014-01-01

    Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

  20. The Regenerative Potential of Parietal Epithelial Cells in Adult Mice

    OpenAIRE

    Berger, Katja; Schulte, Kevin; Boor, Peter; Kuppe, Christoph; van Kuppevelt, Toin H.; Floege, Jürgen; Smeets, Bart; Moeller, Marcus J.

    2014-01-01

    Previously, we showed that some podocytes in juvenile mice are recruited from cells lining Bowman’s capsule, suggesting that parietal epithelial cells (PECs) are a progenitor cell population for podocytes. To investigate whether PECs also replenish podocytes in adult mice, PECs were genetically labeled in an irreversible fashion in 5-week-old mice. No significant increase in labeled podocytes was observed, even after 18 months. To accelerate a potential regenerative mechanism, progressive glo...

  1. Cytotoxic effects of air freshener biocides in lung epithelial cells.

    Science.gov (United States)

    Kwon, Jung-Taek; Lee, Mimi; Seo, Gun-Baek; Kim, Hyun-Mi; Shim, Ilseob; Lee, Doo-Hee; Kim, Taksoo; Seo, Jung Kwan; Kim, Pilje; Choi, Kyunghee

    2013-09-01

    This study evaluated the cytotoxicity of mixtures of citral (CTR) and either benzisothiazolinone (BIT, Mix-CTR-BIT) or triclosan (TCS, Mix-CTR-TCS) in human A549 lung epithelial cells. We investigated the effects of various mix ratios of these common air freshener ingredients on cell viability, cell proliferation, reactive oxygen species (ROS) generation, and DNA damage. Mix-CTR-BIT and Mix-CTR-TCS significantly decreased the viability of lung epithelial cells and inhibited cell growth in a dose-dependent manner. In addition, both mixtures increased ROS generation, compared to that observed in control cells. In particular, cell viability, growth, and morphology were affected upon increase in the proportion of BIT or TCS in the mixture. However, comet analysis showed that treatment of cells with Mix-CTR-BIT or Mix-CTR-TCS did not increase DNA damage. Taken together, these data suggested that increasing the content of biocides in air fresheners might induce cytotoxicity, and that screening these compounds using lung epithelial cells may contribute to hazard assessment.

  2. Renal epithelial cells can release ATP by vesicular fusion

    Directory of Open Access Journals (Sweden)

    Randi G Bjaelde

    2013-09-01

    Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

  3. Loss of Proliferation and Antigen Presentation Activity following Internalization of Polydispersed Carbon Nanotubes by Primary Lung Epithelial Cells

    Science.gov (United States)

    Kumari, Mandavi; Sachar, Sumedha; Saxena, Rajiv K.

    2012-01-01

    Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells. PMID:22384094

  4. Nerve Invasion by Epithelial Cells in Benign Breast Diseases

    Directory of Open Access Journals (Sweden)

    Yu-Jan Chan

    2009-03-01

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

  5. Mesenchymal Stromal Cell-Derived Interleukin-6 Promotes Epithelial-Mesenchymal Transition and Acquisition of Epithelial Stem-Like Cell Properties in Ameloblastoma Epithelial Cells.

    Science.gov (United States)

    Jiang, Chunmiao; Zhang, Qunzhou; Shanti, Rabie M; Shi, Shihong; Chang, Ting-Han; Carrasco, Lee; Alawi, Faizan; Le, Anh D

    2017-09-01

    Epithelial-mesenchymal transition (EMT), a biological process associated with cancer stem-like or cancer-initiating cell formation, contributes to the invasiveness, metastasis, drug resistance, and recurrence of the malignant tumors; it remains to be determined whether similar processes contribute to the pathogenesis and progression of ameloblastoma (AM), a benign but locally invasive odontogenic neoplasm. Here, we demonstrated that EMT- and stem cell-related genes were expressed in the epithelial islands of the most common histologic variant subtype, the follicular AM. Our results revealed elevated interleukin (IL)-6 signals that were differentially expressed in the stromal compartment of the follicular AM. To explore the stromal effect on tumor pathogenesis, we isolated and characterized both mesenchymal stromal cells (AM-MSCs) and epithelial cells (AM-EpiCs) from follicular AM and demonstrated that, in in vitro culture, AM-MSCs secreted a significantly higher level of IL-6 as compared to the counterpart AM-EpiCs. Furthermore, both in vitro and in vivo studies revealed that exogenous and AM-MSC-derived IL-6 induced the expression of EMT- and stem cell-related genes in AM-EpiCs, whereas such effects were significantly abrogated either by a specific inhibitor of STAT3 or ERK1/2, or by knockdown of Slug gene expression. These findings suggest that AM-MSC-derived IL-6 promotes tumor-stem like cell formation by inducing EMT process in AM-EpiCs through STAT3 and ERK1/2-mediated signaling pathways, implying a role in the etiology and progression of the benign but locally invasive neoplasm. Stem Cells 2017;35:2083-2094. © 2017 AlphaMed Press.

  6. Membrane dynamics and the regulation of epithelial cell polarity

    NARCIS (Netherlands)

    van der Wouden, JM; Maier, O; van IJzendoorn, SCD; Hoekstra, D

    2003-01-01

    Plasma membranes of epithelial cells consist of two domains, an apical and a basolateral domain, the surfaces of which differ in composition. The separation of these domains by a tight junction and the fact that specific transport pathways exist for intracellular communication between these domains

  7. Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

    NARCIS (Netherlands)

    Huang, L.; van Loveren, C.; Ling, J.; Wei, X.; Crielaard, W.; Deng, D.M.

    2016-01-01

    Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated

  8. Subtotal ablation of parietal epithelial cells induces crescent formation.

    NARCIS (Netherlands)

    Sicking, E.M.; Fuss, A.; Uhlig, S.; Jirak, P.; Dijkman, H.; Wetzels, J.; Engel, D.R.; Urzynicok, T.; Heidenreich, S.; Kriz, W.; Kurts, C.; Ostendorf, T.; Floege, J.; Smeets, B.; Moeller, M.J.

    2012-01-01

    Parietal epithelial cells (PECs) of the renal glomerulus contribute to the formation of both cellular crescents in rapidly progressive GN and sclerotic lesions in FSGS. Subtotal transgenic ablation of podocytes induces FSGS but the effect of specific ablation of PECs is unknown. Here, we established

  9. Parietal epithelial cells and podocytes in glomerular diseases

    NARCIS (Netherlands)

    Smeets, B.; Moeller, M.J.

    2012-01-01

    In recent years, it has become apparent that parietal epithelial cells (PECs) play an important role within the renal glomerulus, in particular in diseased conditions. In this review, we examine current knowledge about the role of PECs and their interactions with podocytes in development and under

  10. Metabolic cooperativity between epithelial cells and adipocytes of mice

    International Nuclear Information System (INIS)

    Bartley, J.C.; Emerman, J.T.; Bissell, M.J.

    1981-01-01

    We have demonstrated that glycogen and lipid synthesis in adipocytes is modulated by the lactational state and that this modulation in mammary adipocytes requires the presence of the adjacent epithelial cells. Glycogen and lipid synthesis from [ 14 C]glucose was measured in mammary fat pads cleared of epithelium, in abdominal fat pads, and in adipocytes from both sources and from intact mammary gland of mature virgin, pregnant, and lactating mice. Accumulation of glycogen, the activity of glycogen synthase, and the lipogenic rate in abdominal and mammary adipocytes remained high during pregnancy but decreased to insignificant levels by early lactation. The depressant effects of lactation were observed solely in those mammary adipocytes isolated from intact glands. The presence of mammary epithelial cells was also required to effect the stimulated lipogenesis in mammary adipocytes during pregnancy. We conclude that the metabolic activity of adipocytes is modulated both during pregnancy and lactation to channel nutrients to the mammary epithelial cell. The fact that the changes occur in mammary adipocytes only when epithelial cells are present indicates that local as well as systemic factors are operating in these modulations

  11. Parietal epithelial cells: their role in health and disease.

    Science.gov (United States)

    Romagnani, Paola

    2011-01-01

    Parietal epithelial cells of Bowman's capsules were first described by Sir William Bowman in 1842 in his paper On the Structure and Use of the Malpighian Bodies of the Kidney [London, Taylor, 1842], but since then their functions have remained poorly understood. A large body of evidence has recently suggested that parietal epithelial cells represent a reservoir of renal progenitors in adult human kidney which generate novel podocytes during childhood and adolescence, and can regenerate injured podocytes. The discovery that parietal epithelial cells represent a potential source for podocyte regeneration suggests that podocyte injury can be repaired. However, recent results also suggest that an abnormal proliferative response of renal progenitors to podocyte injury can generate hyperplastic glomerular lesions that are observed in crescentic glomerulonephritis and other types of glomerular disorders. Taken together, these results establish an entirely novel view that changes the way of thinking about renal physiology and pathophysiology, and suggest that understanding how self-renewal and fate decision of parietal epithelial cells in response to podocyte injury may be perturbed or modulated will be crucial for obtaining novel tools for prevention and treatment of glomerulosclerosis. Copyright © 2011 S. Karger AG, Basel.

  12. Interrelationship of glyocen metabolism and lactose synthesis in mammary epithelial cells of mice

    Energy Technology Data Exchange (ETDEWEB)

    Emerman, J T; Bartley, J C; Bissell, M J

    1980-01-01

    Glycogen metabolism in mammary epithelial cells was investigated (i) by studying the conversion of glucose into glycogen and other cellular products in these cells from virgin, pregnant and lactating mice and (ii) by assaying the enzymes directly involved with glycogen metabolism. We find that: (1) mammary epithelial cells synthesized glycogen at rates up to over 60% that of the whole gland; (2) the rate of this synthesis was modulated greatly during the reproductive cycle, reaching a peak in late pregnancy and decreasing rapidly at parturition, when abundant synthesis of lactose was initiated. We propose that glycogen bynthesis restricts lactose synthesis during late pregnancy by competing successfully for the shared UDP-glucose pool. The physiological advantage of glycogen accumulation during late pregnancy is discussed.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  16. The fate of epithelial cells in the human large intestine.

    Science.gov (United States)

    Barkla, D H; Gibson, P R

    1999-08-01

    One hundred and forty biopsies of the colon and rectum, collected during routine colonoscopies of 51 patients aged 19 to 74 years, were examined using light microscopy and transmission and scanning electron microscopy. The results indicated that surface epithelial cells undergo apoptosis, passing through fenestrations in the basement membrane to where they enter the lamina propria and are taken up by macrophages; and it is hypothesized that apoptotic cells are carried through the fenestrations on a current of fluid. The study also found that epithelial cells positioned over the crypts are better attached and more robust than those more distant from the crypt opening; and it is further hypothesized that, after reaching the top of the crypts, some goblet cells cease secreting mucus and pass onto the surface compartment of absorptive cells. An unexpected finding was that the lower regions of the crypts commonly contain isolated necrotic colonocytes. Apoptotic cells were rarely observed in the crypt epithelium. The findings of this study support the "recycling" model of epithelial cell death in the surface compartment of the human colon.

  17. Glomerular parietal epithelial cells in kidney physiology, pathology, and repair

    OpenAIRE

    Shankland, Stuart J.; Anders, Hans-Joachim; Romagnani, Paola

    2013-01-01

    Purpose of review We have summarized recently published glomerular parietal epithelial cell (PEC) research, focusing on their roles in glomerular development and physiology, and in certain glomerular diseases. The rationale is that PECs have been largely ignored until the recent availability of cell lineage tracing studies, human and murine PEC culture systems, and potential therapeutic interventions of PECs. Recent findings Several new paradigms involving PECs have emerged demonstrating thei...

  18. The seminiferous epithelial cycle and microanatomy of the koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) testis.

    Science.gov (United States)

    Oishi, Motoharu; Takahashi, Mei; Amasaki, Hajime; Janssen, Tina; Johnston, Stephen D

    2013-03-01

    The koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) are iconic Australian fauna that share a close phylogenetic relationship but there are currently no comparative studies of the seminiferous epithelial cell or testicular microanatomy of either species. Koala and wombat spermatozoa are unusual for marsupials as they possess a curved stream-lined head and lateral neck insertion that superficially is similar to murid spermatozoa; the koala also contains Sertoli cells with crystalloid inclusions that closely resemble the Charcot-Bottcher crystalloids described in human Sertoli cells. Eighteen sexually mature koalas and four sexually mature southern hairy-nosed (SHN) wombats were examined to establish base-line data on quantitative testicular histology. Dynamics of the seminiferous epithelial cycle in the both species consisted of eight stages of cellular association similar to that described in other marsupials. Both species possessed a high proportion of the pre-meiotic (stages VIII, I - III; koala - 62.2 ± 1.7% and SHN wombat - 66.6 ± 2.4%) when compared with post-meiotic stages of the seminiferous cycle. The mean diameters of the seminiferous tubules found in the koalas and the SHN wombats were 227.8 ± 6.1 and 243.5 ± 3.9 μm, respectively. There were differences in testicular histology between the species including the koala possessing (i) a greater proportion of Leydig cells, (ii) larger Sertoli cell nuclei, (iii) crystalloids in the Sertoli cell cytoplasm, (iv) a distinctive acrosomal granule during spermiogenesis and (v) a highly eosinophilic acrosome. An understanding of the seminiferous epithelial cycle and microanatomy of testis is fundamental for documenting normal spermatogenesis and testicular architecture; recent evidence of orchitis and epididymitis associated with natural chlamydial infection in the koala suggest that this species might be useful as an experimental model for understanding Chlamydia

  19. Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

    Science.gov (United States)

    Jijon, H B; Suarez-Lopez, L; Diaz, O E; Das, S; De Calisto, J; Yaffe, M B; Pittet, M J; Mora, J R; Belkaid, Y; Xavier, R J; Villablanca, E J

    2018-05-01

    Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of RA receptor α (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack of RARα resulted in increased KLF4 + goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased Reg3g, reduced luminal bacterial detection, and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

  20. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    International Nuclear Information System (INIS)

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

    1990-01-01

    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

  1. Invasion of Human Oral Epithelial Cells by Prevotella intermedia

    Science.gov (United States)

    Dorn, Brian R.; Leung, K.-P.; Progulske-Fox, Ann

    1998-01-01

    Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion. PMID:9826397

  2. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  3. Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Satoru Yoshida

    Full Text Available BACKGROUND: Application of induced pluripotent stem (iPS cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. METHODOLOGY/PRINCIPAL FINDINGS: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. CONCLUSIONS/SIGNIFICANCE: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.

  4. GM-CSF produced by non-hematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa1,2

    Science.gov (United States)

    Egea, Laia; McAllister, Christopher S.; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F.

    2012-01-01

    GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, and dendritic cell (DC) differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn’s disease in humans and colitis in murine models has been considered mainly to reflect its activity on myeloid cells. We used GM-CSF deficient (GM-CSF−/−) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS) at doses that resulted in little epithelial damage and mucosal ulceration in wild type (WT) mice resulted in marked colon ulceration and delayed ulcer healing in GM-CSF−/− mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF−/− mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF−/− mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Non-hematopoietic cells and not myeloid cells produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury as revealed by bone marrow chimera and DC depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell produced GM-CSF has a novel non-redundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium. PMID:23325885

  5. Trichomonas vaginalis perturbs the junctional complex in epithelial cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Trichomonas vaginalis, a protist parasite of the urogenital tract in humans, is the causative agent of trichomonosis,which in recent years have been associated with the cervical cancer development. In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T. vaginalis and the influence of the iron concentration present in the parasite's culture medium on the interaction effects. Our results show that T. vaginalis adheres to the epithelial cell causing alterations in the junctional complex, such as: (a) a decrease in transepithelial electrical resistance; (b) alteration in the pattern of junctional complex proteins distribution as obseryed for E-cadherin, occludin and ZO-1; and (c) enlargement of the spaces between epithelial cells. These effects were dependent on (a) the degree of the parasite virulence isolate, (b) the iron concentration in the culture medium, and (c) the expression of adhesin proteins on the parasite surface.

  6. Human airway xenograft models of epithelial cell regeneration

    Directory of Open Access Journals (Sweden)

    Puchelle Edith

    2000-10-01

    Full Text Available Abstract Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.

  7. Cigarette smoke alters the secretome of lung epithelial cells.

    Science.gov (United States)

    Mossina, Alessandra; Lukas, Christina; Merl-Pham, Juliane; Uhl, Franziska E; Mutze, Kathrin; Schamberger, Andrea; Staab-Weijnitz, Claudia; Jia, Jie; Yildirim, Ali Ö; Königshoff, Melanie; Hauck, Stefanie M; Eickelberg, Oliver; Meiners, Silke

    2017-01-01

    Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Wnt Signalling in Gastrointestinal Epithelial Stem Cells

    Directory of Open Access Journals (Sweden)

    Dustin J. Flanagan

    2018-03-01

    Full Text Available Wnt signalling regulates several cellular functions including proliferation, differentiation, apoptosis and migration, and is critical for embryonic development. Stem cells are defined by their ability for self-renewal and the ability to be able to give rise to differentiated progeny. Consequently, they are essential for the homeostasis of many organs including the gastrointestinal tract. This review will describe the huge advances in our understanding of how stem cell functions in the gastrointestinal tract are regulated by Wnt signalling, including how deregulated Wnt signalling can hijack these functions to transform cells and lead to cancer.

  9. Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

    Science.gov (United States)

    Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

    2017-01-01

    The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

  10. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Science.gov (United States)

    Viktorinová, Ivana; Henry, Ian; Tomancak, Pavel

    2017-11-01

    Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  11. Osmoregulation of chloride channels in epithelial cells

    NARCIS (Netherlands)

    C.H. Lim (Christina)

    2008-01-01

    markdownabstract__Abstract__ The plasma membrane of mammalian cells is formed by two layers of lipids (lipid bilayer), primarily phospholipids, glycolipids and cholesterol, in which many different proteins are embedded. Phospholipid consists of a glycerol backbone esterified to fatty acids

  12. Epithelial-Mesenchymal Transition in Kidney Tubular Epithelial Cells Induced by Globotriaosylsphingosine and Globotriaosylceramide.

    Directory of Open Access Journals (Sweden)

    Yeo Jin Jeon

    Full Text Available Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A (α-gal A, which results in the deposition of globotriaosylceramide (Gb3 in the vascular endothelium. Globotriaosylsphingosine (lyso-Gb3, a deacylated Gb3, is also increased in the plasma of patients with Fabry disease. Renal fibrosis is a key feature of advanced Fabry disease patients. Therefore, we evaluated the association of Gb3 and lyso-Gb3 accumulation and the epithelial-mesenchymal transition (EMT on tubular epithelial cells of the kidney. In HK2 cells, exogenous treatments of Gb3 and lyso-Gb3 increased the expression of TGF-β, EMT markers (N-cadherin and α-SMA, and phosphorylation of PI3K/AKT, and decreased the expression of E-cadherin. Lyso-Gb3, rather than Gb3, strongly induced EMT in HK2 cells. In the mouse renal mesangial cell line, SV40 MES 13 cells, Gb3 strongly induced phenotype changes. The EMT induced by Gb3 was inhibited by enzyme α-gal A treatment, but EMT induced by lyso-Gb3 was not abrogated by enzyme treatment. However, TGF-β receptor inhibitor (TRI, SB525334 inhibited the activation of TGF-β and EMT markers in HK2 cells with Gb3 and lyso-Gb3 treatments. This study suggested that increased plasma lyso-Gb3 has a crucial role in the development of renal fibrosis through the cell-specific induction of the EMT in Fabry disease, and that TRI treatment, alongside enzyme replacement therapy, could be a potential therapeutic option for patients with Fabry disease.

  13. Inhibition of Wnt Signaling Pathways Impairs Chlamydia trachomatis Infection in Endometrial Epithelial Cells.

    Science.gov (United States)

    Kintner, Jennifer; Moore, Cheryl G; Whittimore, Judy D; Butler, Megan; Hall, Jennifer V

    2017-01-01

    Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures ( p Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis .

  14. Acute damage by naphthalene triggers expression of the neuroendocrine marker PGP9.5 in airway epithelial cells

    DEFF Research Database (Denmark)

    Poulsen, T.T.; Naizhen, X.; Linnoila, R.I.

    2008-01-01

    Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury...... neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27(Kip1), which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27(Kip1) in naphthalene exposed...... and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis Udgivelsesdato: 2008/9/26...

  15. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    Science.gov (United States)

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  16. Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

    Science.gov (United States)

    Ayaki, Masahiko; Yaguchi, Shigeo; Iwasawa, Atsuo; Koide, Ryohei

    2008-08-01

    The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.

  17. TNF-α promotes cell survival through stimulation of K+ channel and NFκB activity in corneal epithelial cells

    International Nuclear Information System (INIS)

    Wang Ling; Reinach, Peter; Lu, Luo

    2005-01-01

    Tumor necrosis factor (TNF-α) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-α also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-α stimulation induced activation of a voltage-gated K + channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-α on downstream events included NFκB nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-α induced increases in p21 expression resulting in partial cell cycle attenuation in the G 1 phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-α-induced K + channel activity effectively prevented NFκB nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-α. In conclusion, TNF-α promotes survival of HCE cells through sequential stimulation of K + channel and NFκB activities. This response to TNF-α is dependent on stimulating K + channel activity because following suppression of K + channel activity TNF-α failed to activate NFκB nuclear translocation and binding to nuclear DNA

  18. Ethane dimethanesulfonate (EDS) perturbs epididymal epithelial cell function in vitro

    International Nuclear Information System (INIS)

    Klinefelter, G.

    1990-01-01

    The formation of sperm granulomas in the epididymis following exposure to EDS, a Leydig cell toxicant, was reported by Cooper and Jackson in 1970. Recent work suggests that EDS may effect the epididymis directly. An in vitro system was developed to determine the nature of any direct effect. The caput epididymis from adult rats was dissected free of connective tissue and small pieces of the tissue were enzymatically digested until plaques of epididymal epithelial cells were obtained. Plaques were cultured on an extracellular matrix gelled on top of a semipermeable filter creating dual-compartment environments. The epithelial cells maintained typical morphology and protein secretion in this culture system for several days. Beginning on day 3, EDS (1 mM) was added to the basal compartment, with or without 35 S-methionine. After 24 hours, 35 S-labelled culture medium was taken from the apical compartment and analyzed by SDS-PAGE and fluorography. EDS caused decreased secretion of several proteins, including a 39 Kd molecule. Interestingly, a 39 Kd protein was also shown to disappear from sperm taken from the caput epididymidis following in vivo exposure to EDS. Unlabelled cultures were fixed and processed for light microscopy. No alterations in morphological integrity were observed. Thus, epididymal epithelial cell function is directly altered by EDS exposure

  19. Serratia marcescens is injurious to intestinal epithelial cells.

    Science.gov (United States)

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

  20. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  1. Infection of Human Fallopian Tube Epithelial Cells with Neisseria gonorrhoeae Protects Cells from Tumor Necrosis Factor Alpha-Induced Apoptosis

    Science.gov (United States)

    Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E.; Christodoulides, Myron; Velasquez, Luis

    2006-01-01

    Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-α antibodies; and (iii) the addition of exogenous TNF-α induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-α-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-α-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

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

    Science.gov (United States)

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  3. Protective effects of trehalose on the corneal epithelial cells.

    Science.gov (United States)

    Aragona, Pasquale; Colosi, Pietro; Rania, Laura; Colosi, Francesca; Pisani, Antonina; Puzzolo, Domenico; Micali, Antonio

    2014-01-01

    Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Twelve patients undergoing laser subepithelial keratomileusis (LASEK) were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. In both trehalose-untreated eyes (TUE) and trehalose-treated eyes (TTE), the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

  4. Protective Effects of Trehalose on the Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Pasquale Aragona

    2014-01-01

    Full Text Available Purpose. Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Methods. Twelve patients undergoing laser subepithelial keratomileusis (LASEK were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. Results. In both trehalose-untreated eyes (TUE and trehalose-treated eyes (TTE, the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Conclusions. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

  5. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine

    NARCIS (Netherlands)

    Klunder, Leon J.; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C. D.

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we

  6. Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.

    Science.gov (United States)

    Sunpaweravong, S; Sunpaweravong, P; Sathitruangsak, C; Mai, S

    2016-05-01

    Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end-to-end fusions. Telomere loss or dysfunction results in breakage-bridge-fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three-dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q-FISH), 3D imaging and 3D analysis of paraffin-embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P-values of P architecture and found no statistically significant differences in any parameter tested between the young and old patients in either the tumor or epithelial cells. The 3D nuclear telomeric signature was able to detect differences in telomere architecture

  7. TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

    Science.gov (United States)

    Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

    2010-01-01

    Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

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

    OpenAIRE

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

    2009-01-01

    The existence of mammary epithelial stem/progenitor cells has been demonstrated in MUC-1-/ ESA+ subpopulations of breast epithelial cells. However, knowledge about the expression and localization in benign and malignant breast lesions is unknown. Using a double-staining immunohistochemistry method, we investigated MUC-1-/ESA+ cells in 10 normal breast tissues, 49 cases with fibrocystic disease, 40 fibroadenomas, 36 invasive ductal carcinomas and the breast cancer ce...

  9. TCDD alters medial epithelial cell differentiation during palatogenesis

    International Nuclear Information System (INIS)

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

    1989-01-01

    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

  10. Isolation, separation, and characterization of epithelial and connective cells from rat palate

    Energy Technology Data Exchange (ETDEWEB)

    Terranova, Victor Paul [Univ. of Rochester, NY (United States)

    1979-01-01

    Epithelial and connective tissue cells were isolated from rat palate by sequential collagenase, hyaluronidase and trypsin digestion of the extracellular matrix. Differences between the two populations were noted with respect to total cell protein, total cell water, proline uptake and incorporation, percent collagen synthesized, effects of parathyroid hormone, metabolism of D-valine and cell density. Basal epithelial cells were subsequently separated from the heterogeneous epithelial cell population on shallow linear density gradients by velocity centrifugation. The type of collagen synthesized by the basal epithelial cells was compared to the type of collagen synthesized by the connective tissue cells by means of labeled amino acid incorporation ratios. Cells isolated from the epithelial and connective tissue were compared. From these studies it can be concluded that epithelial and connective tissue cells can be isolated from rat palate as viable and distinct populations with respect to the biochemical parameters examined. Furthermore, subpopulations can be separated and biochemically characterized.

  11. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xin, Ying [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Lu, Qingxian [Tumor Immunobiology Group, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Li, Qiutang, E-mail: q.li@louisville.edu [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States)

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  12. 14-3-3σ controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    International Nuclear Information System (INIS)

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-01-01

    14-3-3σ (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3σ mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3σ activity in corneal epithelial cells by overexpressing dominative negative 14-3-3σ led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3σ mutant-expressing corneal epithelial cells. We conclude that 14-3-3σ is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  13. Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

    International Nuclear Information System (INIS)

    Moore, Jennifer S.; Rahemtulla, Firoz; Kent, Leigh W.; Hall, Stacy D.; Ikizler, Mine R.; Wright, Peter F.; Nguyen, Huan H.; Jackson, Susan

    2003-01-01

    Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity

  14. A Vitex agnus-castus extract inhibits cell growth and induces apoptosis in prostate epithelial cell lines.

    Science.gov (United States)

    Weisskopf, M; Schaffner, W; Jundt, G; Sulser, T; Wyler, S; Tullberg-Reinert, H

    2005-10-01

    Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

  15. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

    NARCIS (Netherlands)

    Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

    2012-01-01

    The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

  16. Cooperation between epithelial cells demonstrated by potassium transfer

    International Nuclear Information System (INIS)

    Ledbetter, M.L.; Young, G.J.; Wright, E.R.

    1986-01-01

    Junction-mediated communication can be measured in fibroblast cultures by determining the ability of mixed cultures of cells sensitive and resistant to ouabain to concentrate K+ in the presence of ouabain. We now report the extension of this assay procedure to cultured epithelial cells. Hamster kidney (HaK) cells maintain their ability to concentrate K+ in ouabain at levels inhibitory to dog kidney (MDCK) cells. When HaK and MDCK cells were cultured together in ouabain-containing medium, the K+ (measured as 86Rb+) in the mixed population was greater than expected if the cells were not interacting. The degree of enhancement, expressed as index of cooperation, depended on the numbers of cells in the cultures, their opportunity for cell-to-cell contact, and (above a certain permissive level) the concentration of ouabain. As with other cell types, protein synthesis in MDCK cells depends on maintenance of cell K+. Autoradiography of cells incubated with [3H]leucine demonstrated that MDCK cells in ouabain-treated mixed cultures were able to synthesize proteins only when physically adjacent to HaK cells. The transmission of labeled nucleosides among the cells provides independent evidence of the phenomenon of cooperation, probably mediated by gap junctions. This system offers promise for investigation of stimuli modulating junctional communication

  17. Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

    Directory of Open Access Journals (Sweden)

    Fiona L Cousins

    Full Text Available BACKGROUND: In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. METHODOLOGY: A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4 withdrawal; mice received a single injection of bromodeoxyuridine (BrdU 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. PRINCIPAL FINDINGS: Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. CONCLUSIONS/SIGNIFICANCE: These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and

  18. Cellular Homeostasis and Antioxidant Response in Epithelial HT29 Cells on Titania Nanotube Arrays Surface

    Directory of Open Access Journals (Sweden)

    Rabiatul Basria SMN Mydin

    2017-01-01

    Full Text Available Cell growth and proliferative activities on titania nanotube arrays (TNA have raised alerts on genotoxicity risk. Present toxicogenomic approach focused on epithelial HT29 cells with TNA surface. Fledgling cell-TNA interaction has triggered G0/G1 cell cycle arrests and initiates DNA damage surveillance checkpoint, which possibly indicated the cellular stress stimuli. A profound gene regulation was observed to be involved in cellular growth and survival signals such as p53 and AKT expressions. Interestingly, the activation of redox regulator pathways (antioxidant defense was observed through the cascade interactions of GADD45, MYC, CHECK1, and ATR genes. These mechanisms furnish to protect DNA during cellular division from an oxidative challenge, set in motion with XRRC5 and RAD50 genes for DNA damage and repair activities. The cell fate decision on TNA-nanoenvironment has been reported to possibly regulate proliferative activities via expression of p27 and BCL2 tumor suppressor proteins, cogent with SKP2 and BCL2 oncogenic proteins suppression. Findings suggested that epithelial HT29 cells on the surface of TNA may have a positive regulation via cell-homeostasis mechanisms: a careful circadian orchestration between cell proliferation, survival, and death. This nanomolecular knowledge could be beneficial for advanced medical applications such as in nanomedicine and nanotherapeutics.

  19. Proteomic profiling of acrolein adducts in human lung epithelial cells

    Science.gov (United States)

    Spiess, Page C.; Deng, Bin; Hondal, Robert J.; Matthews, Dwight E.; van der Vliet, Albert

    2011-01-01

    Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology. PMID:21704744

  20. Low Temperature Plasma for the Treatment of Epithelial Cancer Cells

    Science.gov (United States)

    Mohades, Soheila

    Biomedical applications of low temperature plasmas (LTP) may lead to a paradigm shift in treating various diseases by conducting fundamental research on the effects of LTP on cells, tissues, organisms (plants, insects, and microorganisms). This is a rapidly growing interdisciplinary research field that involves engineering, physics, life sciences, and chemistry to find novel solutions for urgent medical needs. Effects of different LTP sources have shown the anti-tumor properties of plasma exposure; however, there are still many unknowns about the interaction of plasma with eukaryotic cells which must be elucidated in order to evaluate the practical potential of plasma in cancer treatment. Plasma, the fourth state of matter, is composed of electrons, ions, reactive molecules (radicals and non-radicals), excited species, radiation, and heat. A sufficient dose (time) of plasma exposure can induce death in cancer cells. The plasma pencil is employed to study the anti-tumor properties of this treatment on epithelial cells. The plasma pencil has been previously used for the inactivation of bacteria, destroying amyloid fibrils, and the killing of various cancer cells. Bladder cancer is the 9th leading cause of cancer. In this dissertation, human urinary bladder tissue with the squamous cell carcinoma disease (SCaBER cells) is treated with LTP utilizing two different approaches: direct plasma exposure and Plasma Activated Media (PAM) as an advancement to the treatment. PAM is produced by exposing a liquid cell culture medium to the plasma pencil. Direct LTP treatment of cancer cells indicates a dose-dependent killing effect at post-treatment times. Similarly, PAM treatment shows an anti-cancer effect by inducing substantial cell death. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have an important role in the biomedical effects of LTP treatment. This study demonstrates the capability of the plasma pencil to transport ROS/RNS into cell culture media

  1. ATP7B detoxifies silver in ciliated airway epithelial cells

    International Nuclear Information System (INIS)

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

    2010-01-01

    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 -/- 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 + /Cu + transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

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

    Science.gov (United States)

    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...

  3. ATM suppresses SATB1-induced malignant progression in breast epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ellen Ordinario

    Full Text Available SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2, but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.

  4. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  5. Inhibition of EV71 by curcumin in intestinal epithelial cells.

    Science.gov (United States)

    Huang, Hsing-I; Chio, Chi-Chong; Lin, Jhao-Yin

    2018-01-01

    EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.

  6. Inhibition of EV71 by curcumin in intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hsing-I Huang

    Full Text Available EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6, an active ingredient of turmeric (Curcuma longa Linn with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK signaling pathways is not involved. We found that protein kinase C delta (PKCδ plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.

  7. Inhibition of EV71 by curcumin in intestinal epithelial cells

    Science.gov (United States)

    Chio, Chi-Chong; Lin, Jhao-Yin

    2018-01-01

    EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections. PMID:29370243

  8. Effect of taurine on advanced glycation end products-induced hypertrophy in renal tubular epithelial cells

    International Nuclear Information System (INIS)

    Huang, J.-S.; Chuang, L.-Y.; Guh, J.-Y.; Yang, Y.-L.; Hsu, M.-S.

    2008-01-01

    Mounting evidence indicates that advanced glycation end products (AGE) play a major role in the development of diabetic nephropathy (DN). Taurine is a well documented antioxidant agent. To explore whether taurine was linked to altered AGE-mediated renal tubulointerstitial fibrosis in DN, we examined the molecular mechanisms of taurine responsible for inhibition of AGE-induced hypertrophy in renal tubular epithelial cells. We found that AGE (but not non-glycated BSA) caused inhibition of cellular mitogenesis rather than cell death by either necrosis or apoptosis. There were no changes in caspase 3 activity, bcl-2 protein expression, and mitochondrial cytochrome c release in BSA, AGE, or the antioxidant taurine treatments in these cells. AGE-induced the Raf-1/extracellular signal-regulated kinase (ERK) activation was markedly blocked by taurine. Furthermore, taurine, the Raf-1 kinase inhibitor GW5074, and the ERK kinase inhibitor PD98059 may have the ability to induce cellular proliferation and cell cycle progression from AGE-treated cells. The ability of taurine, GW5074, or PD98059 to inhibit AGE-induced hypertrophy was verified by the observation that it significantly decreased cell size, cellular hypertrophy index, and protein levels of RAGE, p27 Kip1 , collagen IV, and fibronectin. The results obtained in this study suggest that taurine may serve as the potential anti-fibrotic activity in DN through mechanism dependent of its Raf-1/ERK inactivation in AGE-induced hypertrophy in renal tubular epithelial cells

  9. Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation

    Directory of Open Access Journals (Sweden)

    Andrea C. Lei-Leston

    2017-09-01

    Full Text Available Pattern recognition receptors (PRR, such as NOD-like receptors (NLRs, sense conserved microbial signatures, and host danger signals leading to the coordination of appropriate immune responses. Upon activation, a subset of NLR initiate the assembly of a multimeric protein complex known as the inflammasome, which processes pro-inflammatory cytokines and mediates a specialized form of cell death known as pyroptosis. The identification of inflammasome-associated genes as inflammatory bowel disease susceptibility genes implicates a role for the inflammasome in intestinal inflammation. Despite the fact that the functional importance of inflammasomes within immune cells has been well established, the contribution of inflammasome expression in non-hematopoietic cells remains comparatively understudied. Given that intestinal epithelial cells (IEC act as a barrier between the host and the intestinal microbiota, inflammasome expression by these cells is likely important for intestinal immune homeostasis. Accumulating evidence suggests that the inflammasome plays a key role in shaping epithelial responses at the host–lumen interface with many inflammasome components highly expressed by IEC. Recent studies have exposed functional roles of IEC inflammasomes in mucosal immune defense, inflammation, and tumorigenesis. In this review, we present the main features of the predominant inflammasomes and their effector mechanisms contributing to intestinal homeostasis and inflammation. We also discuss existing controversies in the field and open questions related to their implications in disease. A comprehensive understanding of the molecular basis of intestinal inflammasome signaling could hold therapeutic potential for clinical translation.

  10. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

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    Shuxian Jiang

    2010-03-01

    Full Text Available Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  11. Colonic epithelial cell activation and the paradoxical effects of butyrate.

    Science.gov (United States)

    Gibson, P R; Rosella, O; Wilson, A J; Mariadason, J M; Rickard, K; Byron, K; Barkla, D H

    1999-04-01

    Butyrate may have paradoxical effects on epithelial cells of similar origin. This study aimed to examine the hypothesis that one mechanism that dictates a cell's response to butyrate is its state of activation. First, the responses to 24 h exposure to butyrate (1-2 mM) of normal and neoplastic human colonic epithelial cells activated by their isolation and primary culture, and of colon cancer cell lines, LIM1215 and Caco-2, were examined. In primary cultures of normal and cancer cells, butyrate had no effect on alkaline phosphatase activities but significantly suppressed urokinase receptor expression by a mean +/- SEM of 30 +/- 12% and 36 +/- 9%, respectively. Interleukin-8 secretion was suppressed by 44 +/- 7% in normal cells (P 50%, urokinase receptor expression >2-fold and interleukin-8 secretion >3-fold in response to butyrate. Secondly, the effect of butyrate on Caco-2 cells was examined with or without prior exposure to a specific activating stimulus [tumour necrosis factor alpha (TNF alpha)]. Interleukin-8 secretion increased by 145 +/- 23% and 132 +/- 17% on 24 h exposure to 2 mM butyrate or 0.1 microM TNF alpha alone, respectively. However, in cells pre-treated with TNF alpha, butyrate significantly inhibited secretion by 34 +/- 7% below unstimulated levels. The response to butyrate of urokinase receptor, whose expression was not stimulated by TNF alpha, was unchanged. These effects were mimicked by trichostatin A, an inhibitor of histone deacetylase, suggesting that butyrate's paradoxical effects may have been operating by the same mechanism. In conclusion, some of the paradoxical effects of butyrate do not appear to represent inherent differences between normal and transformed cells. Rather, the response may be determined by the state of activation of the cells.

  12. Immunoregulation by airway epithelial cells (AECs against respiratory virus infection

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    Yan YAN

    2017-11-01

    Full Text Available The respiratory tract is primary contact site of the body and environment, and it is ventilated by 10-20 thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbes, which contain the disease-causing pathogens. Airway epithelial cells (AECs are known to have innate sensor functions, which are similar to the "professional" immune cells, such as alveolar macrophage and sub- or intra-epithelial dendritic cells (DCs. Thus AECs are able to detect invading microbial danger including different types of respiratory viruses, and mount a potent host response, for example, activating type Ⅰ interferon signaling pathway genes. To avoid chronic inflammation and maintain the immunological homeostasis, the pulmonary system has developed intrinsic mechanisms to control local immune responses. Most recently, the role of AECs in control of local immunity has gained much attention, as 1 AECs express the pattern recognition receptors (PRRs, such as Toll-like receptors, retinoic acid inducible gene Ⅰ (RIG-I-like receptor, and so on, thus AECs are equipped to participate in innate detection of microbial encounter; 2 To keep immunological homeostasis in the respiratory tract, AECs behave not only as innate immune sensors but also as immune modulators in parallel, through modulating the sensitivity of innate immune sensing of both AECs per se and sub- or intra-epithelial immune cells; 3 Loss of modularity capacity of AECs might be involved in the development of chronic airway diseases. In present review, how the AECs act will be intensively discussed in response to respiratory viruses and modulate the local immunity through cis- and trans-factors (direct and indirect factors, as well as the consequence of impairment of this control of local immunity, in the development and exacerbation of airway diseases, such as acute and chronic rhinosinusitis. DOI: 10.11855/j.issn.0577-7402.2017.10.02

  13. Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway

    International Nuclear Information System (INIS)

    Gao, Rundi; Chen, Ruilin; Cao, Yu; Wang, Yuan; Song, Kang; Zhang, Ya; Yang, Junchao

    2017-01-01

    Pulmonary fibrosis is characterized by the destruction of lung tissue architecture and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a component of Chinese herb that has been reported to be medicament on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been established yet. In the present study, we investigated the hypothesis that Emodin plays an inhibitory role in TGF-β1 induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cell, and Emodin exerts its effect through the Notch signaling pathway. Emodin inhibits the proliferation of Rat alveolar type II epithelial cells RLE-6TN in a concentration-dependent manner; reduces the expression of Collagen I, α-SMA and Vimentin, promotes the expression of E-cadherin. Moreover, Emodin could regulate the expression patterns of the Notch signaling pathway-related factors and reduce the Notch-1 nucleus translocation. Knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. In conclusion, the data of the present study suggests that Emodin suppresses TGF-β1-induced EMT in alveolar epithelial cells through Notch signaling pathway and shows the potential to be effective in the treatment of pulmonary fibrosis. - Highlights: • Emodin inhibits TGF-β1-induced EMT in alveolar epithelial cells. • Emodin regulates the expression patterns of the Notch signaling pathway-related factors. • Emodin inhibits TGF-β1-induced Notch-1 nucleus translocation and activation.

  14. Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Rundi; Chen, Ruilin; Cao, Yu [Department of Respiration, The First Affiliated Hospital of Zhejiang Chinese Medicine University, NO. 56, Youdian Road, Shangcheng District, Hangzhou, Zhejiang Province 310006 (China); Wang, Yuan [Department of Pulmonary Function, The First Affiliated Hospital of Zhejiang Chinese Medicine University, NO. 56, Youdian Road, Shangcheng District, Hangzhou, Zhejiang Province 310006 (China); Song, Kang [Department of Respiration, The First Affiliated Hospital of Zhejiang Chinese Medicine University, NO. 56, Youdian Road, Shangcheng District, Hangzhou, Zhejiang Province 310006 (China); Zhang, Ya [Zhejiang Chinese Medicine University, No. 548, Binwen Road, Binjiang District, Hangzhou, Zhejiang Province 310006 (China); Yang, Junchao, E-mail: yangjunchaozj@zcmu.edu.cn [Department of Respiration, The First Affiliated Hospital of Zhejiang Chinese Medicine University, NO. 56, Youdian Road, Shangcheng District, Hangzhou, Zhejiang Province 310006 (China)

    2017-03-01

    Pulmonary fibrosis is characterized by the destruction of lung tissue architecture and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a component of Chinese herb that has been reported to be medicament on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been established yet. In the present study, we investigated the hypothesis that Emodin plays an inhibitory role in TGF-β1 induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cell, and Emodin exerts its effect through the Notch signaling pathway. Emodin inhibits the proliferation of Rat alveolar type II epithelial cells RLE-6TN in a concentration-dependent manner; reduces the expression of Collagen I, α-SMA and Vimentin, promotes the expression of E-cadherin. Moreover, Emodin could regulate the expression patterns of the Notch signaling pathway-related factors and reduce the Notch-1 nucleus translocation. Knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. In conclusion, the data of the present study suggests that Emodin suppresses TGF-β1-induced EMT in alveolar epithelial cells through Notch signaling pathway and shows the potential to be effective in the treatment of pulmonary fibrosis. - Highlights: • Emodin inhibits TGF-β1-induced EMT in alveolar epithelial cells. • Emodin regulates the expression patterns of the Notch signaling pathway-related factors. • Emodin inhibits TGF-β1-induced Notch-1 nucleus translocation and activation.

  15. Aquaporin 2 promotes cell migration and epithelial morphogenesis.

    Science.gov (United States)

    Chen, Ying; Rice, William; Gu, Zhizhan; Li, Jian; Huang, Jianmin; Brenner, Michael B; Van Hoek, Alfred; Xiong, Jianping; Gundersen, Gregg G; Norman, Jim C; Hsu, Victor W; Fenton, Robert A; Brown, Dennis; Lu, Hua A Jenny

    2012-09-01

    The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin β1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin β1 by mutating the RGD motif led to reduced endocytosis, retention of integrin β1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin β1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.

  16. Aging effects on intestinal homeostasis associated with expansion and dysfunction of intestinal epithelial stem cells.

    Science.gov (United States)

    Moorefield, Emily C; Andres, Sarah F; Blue, R Eric; Van Landeghem, Laurianne; Mah, Amanda T; Santoro, M Agostina; Ding, Shengli

    2017-08-29

    Intestinal epithelial stem cells (IESCs) are critical to maintain intestinal epithelial function and homeostasis. We tested the hypothesis that aging promotes IESC dysfunction using old (18-22 months) and young (2-4 month) Sox9-EGFP IESC reporter mice. Different levels of Sox9-EGFP permit analyses of active IESC (Sox9-EGFP Low ), activatable reserve IESC and enteroendocrine cells (Sox9-EGFP High ), Sox9-EGFP Sublow progenitors, and Sox9-EGFP Negative differentiated lineages. Crypt-villus morphology, cellular composition and apoptosis were measured by histology. IESC function was assessed by crypt culture, and proliferation by flow cytometry and histology. Main findings were confirmed in Lgr5-EGFP and Lgr5-LacZ mice. Aging-associated gene expression changes were analyzed by Fluidigm mRNA profiling. Crypts culture from old mice yielded fewer and less complex enteroids. Histology revealed increased villus height and Paneth cells per crypt in old mice. Old mice showed increased numbers and hyperproliferation of Sox9-EGFP Low IESC and Sox9-EGFP High cells. Cleaved caspase-3 staining demonstrated increased apoptotic cells in crypts and villi of old mice. Gene expression profiling revealed aging-associated changes in mRNAs associated with cell cycle, oxidative stress and apoptosis specifically in IESC. These findings provide new, direct evidence for aging associated IESC dysfunction, and define potential biomarkers and targets for translational studies to assess and maintain IESC function during aging.

  17. Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

    Science.gov (United States)

    Nakajima, Ryota; Takeda, Shizu

    2014-01-01

    The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

    International Nuclear Information System (INIS)

    Roberts, Joan E.; Wielgus, Albert R.; Boyes, William K.; Andley, Usha; Chignell, Colin F.

    2008-01-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C 60 (OH) 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 60 (OH) 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 μM. Exposure to either UVA or visible light in the presence of > 5 μM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 μM 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 α-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 60 (OH) 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.

    Directory of Open Access Journals (Sweden)

    Han Hu

    Full Text Available 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 beta-casein (CSN2, acetyl-CoA carboxylase-alpha (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.

  20. Implantation of Induced Pluripotent Stem Cell-Derived Tracheal Epithelial Cells.

    Science.gov (United States)

    Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Nakamura, Ryosuke; Otsuki, Koshi; Murono, Shigeyuki; Omori, Koichi

    2017-07-01

    Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects. Red fluorescent tdTomato protein was electroporated into mouse iPSCs to produce tdTomato-labeled iPSCs. Embryoid bodies derived from these iPSCs were then cultured in differentiation medium supplemented with growth factors, followed by culture on air-liquid interfaces for further differentiation into tracheal epithelium. The cells were implanted with artificial tracheas into nude rats with tracheal defects on day 26 of cultivation. On day 7 after implantation, the tracheas were exposed and examined histologically. Tracheal epithelial tissue derived from tdTomato-labeled iPSCs survived in the tracheal defects. Moreover, immunochemical analyses showed that differentiated tissues had epithelial structures similar to those of proximal tracheal tissues. After differentiation from iPSCs, tracheal epithelial tissues survived in rat bodies, warranting the use of iPSCs for epithelial regeneration in tracheal defects.

  1. Cellular and Nuclear Alignment Analysis for Determining Epithelial Cell Chirality

    Science.gov (United States)

    Raymond, Michael J.; Ray, Poulomi; Kaur, Gurleen; Singh, Ajay V.; Wan, Leo Q.

    2015-01-01

    Left-right (LR) asymmetry is a biologically conserved property in living organisms that can be observed in the asymmetrical arrangement of organs and tissues and in tissue morphogenesis, such as the directional looping of the gastrointestinal tract and heart. The expression of LR asymmetry in embryonic tissues can be appreciated in biased cell alignment. Previously an in vitro chirality assay was reported by patterning multiple cells on microscale defined geometries and quantified the cell phenotype–dependent LR asymmetry, or cell chirality. However, morphology and chirality of individual cells on micropatterned surfaces has not been well characterized. Here, a Python-based algorithm was developed to identify and quantify immunofluorescence stained individual epithelial cells on multicellular patterns. This approach not only produces results similar to the image intensity gradient-based method reported previously, but also can capture properties of single cells such as area and aspect ratio. We also found that cell nuclei exhibited biased alignment. Around 35% cells were misaligned and were typically smaller and less elongated. This new imaging analysis approach is an effective tool for measuring single cell chirality inside multicellular structures and can potentially help unveil biophysical mechanisms underlying cellular chiral bias both in vitro and in vivo. PMID:26294010

  2. Ouabain Increases Gap Junctional Communication in Epithelial Cells

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    Arturo Ponce

    2014-11-01

    Full Text Available Background/Aims: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC. Methods: We employed two different approaches: 1 analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2 measurement of the electrical capacitance. Results: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. Conclusion: Ouabain 10 nM increases GJC in MDCK cells.

  3. [Inhibitory effect of diclofenac sodium on the proliferation of rabbit corneal epithelial cells in vitro].

    Science.gov (United States)

    Wu, Ningling; Du, Zhiyu

    2010-11-01

    To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. The fresh rabbit cornea was cultured to obtain the primary RCECs, and RCECs of passage 2 were used in this research. The cells were divided into experimental groups, the cells in which were incubated with different concentrations (18.18, 27.27, 36.36, 45.45, 54.55 μg/ml) of diclofenac sodium, and control group. The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay 24, 48 and 72 h after incubation. While the RCECs were divided into experimental groups, the cells in which were incubated with 9 and 12.5 μg/ml diclofenac sodium, and control group. The cell cycle and apoptotic rate were observed by flow cytometer. MTT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs, and the inhibition rate was increasing along with the increasing concentration of diclofenac sodium and the incubation time(Pdiclofenac sodium, the cells in G0/G1 phase were obviously increased, and the apoptosis cusp and apoptotic rate were increased. Diclofenac sodium exerts significant inhibitory effect on RCECs in a dosage-dependent manner, and it may function by inducing cell apoptosis and ceasing cell cycles.

  4. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    Directory of Open Access Journals (Sweden)

    Llobet-Brossa Enrique

    2009-08-01

    Full Text Available Abstract 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.

  5. Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation

    Science.gov (United States)

    Damodarasamy, Mamatha; Vernon, Robert B.; Chan, Christina K.; Plymate, Stephen R.; Wight, Thomas N.

    2015-01-01

    The extracellular matrix (ECM) of the prostate, which is comprised primarily of collagen, becomes increasingly disorganized with age, a property that may influence the development of hyperplasia and cancer. Collageous ECM extracted from the tails of aged mice exhibits many characteristics of collagen in aged tissues, including the prostate. When polymerized into a 3-dimensional (3D) gel, these collagen extracts can serve as models for the study of specific cell-ECM interactions. In the present study, we examined the behaviors of human prostatic epithelial cell lines representing normal prostate epithelial cells (PEC), benign prostatic hyperplasia (BPH-1), and adenocarcinoma (LNCaP) cultured in contact with 3D gels made from collagen extracts of young and aged mice. We found that proliferation of PEC, BPH-1, and LNCaP cells were all increased by culture on aged collagen gels relative to young collagen gels. In examining age-associated differences in the composition of the collagen extracts, we found that aged and young collagen had a similar amount of several collagen-associated ECM components, but aged collagen had a much greater content of the glycosaminoglycan hyaluronan (HA) than young collagen. The addition of HA (of similar size and concentration to that found in aged collagen extracts) to cells placed in young collagen elicited significantly increased proliferation in BPH-1 cells, but not in PEC or LNCaP cells, relative to controls not exposed to HA. Of note, histochemical analyses of human prostatic tissues showed significantly higher expression of HA in BPH and prostate cancer stroma relative to stroma of normal prostate. Collectively, these results suggest that changes in ECM involving increased levels of HA contribute to the growth of prostatic epithelium with aging. PMID:25124870

  6. Epithelial Cell-Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia.

    Science.gov (United States)

    Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A; Zabinski, Mary C; Yuen, Constance K; Lung, Wing Yi; Gower, Adam C; Belkina, Anna C; Ramirez, Maria I; Deng, Jane C; Quinton, Lee J; Jones, Matthew R; Mizgerd, Joseph P

    2016-09-01

    Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6G(bright)CD11b(bright) neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia.

  7. Epigenetic dynamics across the cell cycle

    DEFF Research Database (Denmark)

    Kheir, Tony Bou; Lund, Anders H.

    2010-01-01

    Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

  8. CXCL9 Regulates TGF-β1-Induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells.

    Science.gov (United States)

    O'Beirne, Sarah L; Walsh, Sinead M; Fabre, Aurélie; Reviriego, Carlota; Worrell, Julie C; Counihan, Ian P; Lumsden, Robert V; Cramton-Barnes, Jennifer; Belperio, John A; Donnelly, Seamas C; Boylan, Denise; Marchal-Sommé, Joëlle; Kane, Rosemary; Keane, Michael P

    2015-09-15

    Epithelial to mesenchymal cell transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial marker thyroid transcription factor-1 and the mesenchymal marker α-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-β1 and CXCL9, with Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-β1-induced EMT. A decrease in TGF-β1-induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-β1-induced EMT. Copyright © 2015 by The American Association of Immunologists, Inc.

  9. γδ T cells in homeostasis and host defence of epithelial barrier tissues

    DEFF Research Database (Denmark)

    Nielsen, Morten M.; Witherden, Deborah A.; Havran, Wendy L.

    2017-01-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue...... homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body — namely, the epidermis and the intestine — and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity...

  10. Production of arachidonic and linoleic acid metabolites by guinea pig tracheal epithelial cells

    International Nuclear Information System (INIS)

    Oosthuizen, M.J.; Engels, F.; Van Esch, B.; Henricks, P.A.; Nijkamp, F.P.

    1990-01-01

    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

  11. Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia.

    Science.gov (United States)

    Mebratu, Yohannes A; Schwalm, Kurt; Smith, Kevin R; Schuyler, Mark; Tesfaigzi, Yohannes

    2011-06-01

    Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. We screened for dysregulated expression of the Bcl-2 family members. We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.

  12. Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells.

    Science.gov (United States)

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

    Science.gov (United States)

    Armitage, Mark

    Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or

  14. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  15. Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells

    International Nuclear Information System (INIS)

    Perkins, Timothy N.; Dentener, Mieke A.; Stassen, Frank R.; Rohde, Gernot G.; Mossman, Brooke T.; Wouters, Emiel F.M.; Reynaert, Niki L.

    2016-01-01

    Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. - Highlights: • Pathway analysis reveals silica-induced WNT-signaling in lung epithelial cells. • Silica-induced canonical WNT-signaling is mediated by autocrine/paracrine signals. • Crystalline silica decreases non-canonical WNT

  16. Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, Timothy N.; Dentener, Mieke A. [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Stassen, Frank R. [Department of Medical Microbiology, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Rohde, Gernot G. [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Mossman, Brooke T. [Department of Pathology, University of Vermont College of Medicine, Burlington, VT (United States); Wouters, Emiel F.M. [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Reynaert, Niki L., E-mail: n.reynaert@maastrichtuniversity.nl [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands)

    2016-06-15

    Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. - Highlights: • Pathway analysis reveals silica-induced WNT-signaling in lung epithelial cells. • Silica-induced canonical WNT-signaling is mediated by autocrine/paracrine signals. • Crystalline silica decreases non-canonical WNT

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

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

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

  18. Equine tracheal epithelial membrane strips - An alternate method for examining epithelial cell arachidonic acid metabolism

    International Nuclear Information System (INIS)

    Gray, P.R.; Derksen, F.J.; Robinson, N.E.; Peter-Golden, M.L.

    1990-01-01

    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 (≤12 x 1.5 cm) of pseudostratified columnar epithelium attached to a layer of elastic tissue 30-100 μm thick. Epithelial strips (1.2 x 0.5 cm) were attached to plexiglass rods and incubated with [ 3 H]arachidonic acid in M199 medium (0.5 μCi/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 μM calcium ionophore A23187. The extracted supernatant was processed using HPLC, resulting in peaks of radioactivity that co-eluted with authentic PGE 2 , PGF 2 α, and 12-HETE standards. The greatest activity corresponded to the PGE 2 and PGF 2 α standards, which is a similar pattern to that reported for cultured human tracheal epithelium

  19. Follicular dermal papilla structures by organization of epithelial and mesenchymal cells in interfacial polyelectrolyte complex fibers.

    Science.gov (United States)

    Lim, Tze Chiun; Leong, Meng Fatt; Lu, Hongfang; Du, Chan; Gao, Shujun; Wan, Andrew C A; Ying, Jackie Y

    2013-09-01

    The hair follicle is a regenerating organ that produces a new hair shaft during each growth cycle. Development and cycling of the hair follicle is governed by interactions between the epithelial and mesenchymal components. Therefore, development of an engineered 3D hair follicle would be useful for studying these interactions to identify strategies for treatment of hair loss. We have developed a technique suitable for assembly of different cell types in close proximity in fibrous hydrogel scaffolds with resolutions of ∼50 μm. By assembly of dermal papilla (DP) and keratinocytes, structures similar to the native hair bulb arrangement are formed. Gene expression of these constructs showed up-regulation of molecules involved in epithelial-mesenchymal interactions of the hair follicle. Implantation of the follicular structures in SCID mice led to the formation of hair follicle-like structures, thus demonstrating their hair inductive ability. The transparency of the fiber matrix and the small dimensions of the follicular structures allowed the direct quantitation of DP cell proliferation by confocal microscopy, clearly illustrating the promoting or inhibitory effects of hair growth regulating agents. Collectively, our results suggested a promising application of these 3D engineered follicular structures for in vitro screening and testing of drugs for hair growth therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. NoxO1 Controls Proliferation of Colon Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Franziska Moll

    2018-05-01

    Full Text Available AimReactive oxygen species (ROS produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.ResultsNoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.ConclusionNoxO1 affects colon epithelium homeostasis and prevents inflammation.

  1. Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

    Directory of Open Access Journals (Sweden)

    Takashi Kawaguchi

    1999-01-01

    Full Text Available This study was designed to examine the effects of adrenomedullin (AM on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP, cyclic guanosine monophosphate (cGMP, prostaglandin E2 (PGE2, and stable end-products of nitric oxide were assayed. Adrenomedullin (10−6 mol/L stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10−5 mol/L significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10−8 – 10−6 mol/L did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

  2. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

    Science.gov (United States)

    Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

    2014-01-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

  3. CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

    Science.gov (United States)

    Yu, Shiguang; Fang, Yujiang; Sharav, Tumenjargal; Sharp, Gordon C; Braley-Mullen, Helen

    2011-02-15

    CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.

  4. Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

    Science.gov (United States)

    Li, Ying; Bentzley, Catherine M; Tarloff, Joan B

    2005-04-01

    Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations

  5. A method for isolating identifying and culturing of rat trachea-bronchia epithelial cells

    International Nuclear Information System (INIS)

    Cui Fengmei; Su Shibiao; Nie Jihua; Li Bingyan; Tong Jian

    2005-01-01

    Objective: To explore a method for isolating identifying and culturing the rat trachea-bronchia epithelial cells. Methods: The rat trachea-bronchia epithelial cells were isolated by digestion with pronase and brushing with cell brush, identified using confocul and cultured in entire F12 media with no serum. Results: With this method, cells in high purity and high viability could be obtained, and about 10 6 cells per rat. The cells grow well in entire F12 media with no serum. Conclusion: The method is useful for isolating rate trachea-bronchia epithelial cells and the entire F12 media with no serum is effective for culturing. (authors)

  6. Centriole movements in mammalian epithelial cells during cytokinesis

    Directory of Open Access Journals (Sweden)

    Tanke Hans J

    2010-05-01

    Full Text Available Abstract Background In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules. Results Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1 along the nuclear envelope, 2 irregular, and 3 along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent. Conclusions These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.

  7. Effect of Formaldehyde on Human Middle Ear Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Shin Hye Kim

    2018-01-01

    Full Text Available Formaldehyde (FA is a familiar indoor air pollutant found in everything from cosmetics to clothing, but its impact on the middle ear is unknown. This study investigated whether FA causes cytotoxicity, inflammation, or induction of apoptosis in human middle ear epithelial cells (HMEECs. Cell viability was investigated using the trypan blue assay and a cell counting kit (CCK-8 in HMEECs treated with FA for 4 or 24 h. The expression of genes encoding the inflammatory cytokine tumor necrosis factor alpha (TNF-α and mucin (MUC5AC was analyzed using RT-PCR. Activation of the apoptosis pathway was determined by measuring mitochondrial membrane potential (MMP, cytochrome oxidase, caspase-9/Mch6/Apaf 3, and Caspase-Glo® 3/7 activities. The CCK-8 assay and trypan blue assay results showed a reduction in cell viability in FA-treated HMEECs. FA also increased the cellular expression of TNF-α and MUC5AC and reduced the activities of MMP and cytochrome oxidase. Caspase-9 activity increased in cells stimulated for 4 h, as well as caspase-3/7 activity in cells stimulated for 24 h. The decreased cell viability, the induction of inflammation and mucin gene expression, and the activation of the apoptosis pathway together indicate a link between environmental FA exposure and the development of otitis media.

  8. The regenerative potential of parietal epithelial cells in adult mice.

    Science.gov (United States)

    Berger, Katja; Schulte, Kevin; Boor, Peter; Kuppe, Christoph; van Kuppevelt, Toin H; Floege, Jürgen; Smeets, Bart; Moeller, Marcus J

    2014-04-01

    Previously, we showed that some podocytes in juvenile mice are recruited from cells lining Bowman's capsule, suggesting that parietal epithelial cells (PECs) are a progenitor cell population for podocytes. To investigate whether PECs also replenish podocytes in adult mice, PECs were genetically labeled in an irreversible fashion in 5-week-old mice. No significant increase in labeled podocytes was observed, even after 18 months. To accelerate a potential regenerative mechanism, progressive glomerular hypertrophy was induced by progressive partial nephrectomies. Again, no significant podocyte replenishment was observed. Rather, labeled PECs exclusively invaded segments of the tuft affected by glomerulosclerosis, consistent with our previous findings. We next reassessed PEC recruitment in juvenile mice using a different reporter mouse and confirmed significant recruitment of labeled PECs onto the glomerular tuft. Moreover, some labeled cells on Bowman's capsule expressed podocyte markers, and cells on Bowman's capsule were also directly labeled in juvenile podocyte-specific Pod-rtTA transgenic mice. In 6-week-old mice, however, cells on Bowman's capsule no longer expressed podocyte-specific markers. Similarly, in human kidneys, some cells on Bowman's capsule expressed the podocyte marker synaptopodin from 2 weeks to 2 years of age but not at 7 years of age. In summary, podocyte regeneration from PECs could not be detected in aging mice or models of glomerular hypertrophy. We propose that a small fraction of committed podocytes reside on Bowman's capsule close to the vascular stalk and are recruited onto the glomerular tuft during infancy to adolescence in mice and humans.

  9. Depleted uranium induces neoplastic transformation in human lung epithelial cells.

    Science.gov (United States)

    Xie, Hong; LaCerte, Carolyne; Thompson, W Douglas; Wise, John Pierce

    2010-02-15

    Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and noncombatants is frequent and widespread. Previous studies have shown that DU has both chemical and radiological toxicity and that the primary route of exposure of DU to humans is through inhalation and ingestion. However, there is limited research information on the potential carcinogenicity of DU in human bronchial cells. Accordingly, we determined the neoplastic transforming ability of particulate DU to human bronchial epithelial cells (BEP2D). We observed the loss of contact inhibition and anchorage independent growth in cells exposed to DU after 24 h. We also characterized these DU-induced transformed cell lines and found that 40% of the cell lines exhibit alterations in plating efficiency and no significant changes in the cytotoxic response to DU. Cytogenetic analyses showed that 53% of the DU-transformed cell lines possess a hypodiploid phenotype. These data indicate that human bronchial cells are transformed by DU and exhibit significant chromosome instability consistent with a neoplastic phenotype.

  10. The Inside Story of Shigella Invasion of Intestinal Epithelial Cells

    Science.gov (United States)

    Carayol, Nathalie; Tran Van Nhieu, Guy

    2013-01-01

    As opposed to other invasive pathogens that reside into host cells in a parasitic mode, Shigella, the causative agent of bacillary dysentery, invades the colonic mucosa but does not penetrate further to survive into deeper tissues. Instead, Shigella invades, replicates, and disseminates within the colonic mucosa. Bacterial invasion and spreading in intestinal epithelium lead to the elicitation of inflammatory responses responsible for the tissue destruction and shedding in the environment for further infection of other hosts. In this article, we highlight specific features of the Shigella arsenal of virulence determinants injected by a type III secretion apparatus (T3SA) that point to the targeting of intestinal epithelial cells as a discrete route of invasion during the initial event of the infectious process. PMID:24086068

  11. Regulation of potassium transport in human lens epithelial cells.

    Science.gov (United States)

    Lauf, Peter K; Warwar, Ronald; Brown, Thomas L; Adragna, Norma C

    2006-01-01

    The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed 100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.

  12. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine.

    Science.gov (United States)

    Klunder, Leon J; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C D

    2017-07-05

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we highlight recent advances with regard to the molecular mechanisms of cell polarity-controlled epithelial homeostasis and immunity in the human intestine. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  13. Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

    OpenAIRE

    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 sequ...

  14. Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

    Science.gov (United States)

    Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

    1984-11-01

    During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

  15. Effect of Lead on Human Middle Ear Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Shin Hye Kim

    2018-01-01

    Full Text Available Lead is a ubiquitous metal in the environment, but no studies have examined lead toxicity on the middle ear. Here, we investigated lead toxicity and its mechanism in human middle ear epithelial cells (HMEECs. Moreover, we investigated the protective effects of amniotic membrane extract (AME and chorionic membrane extract (CME against lead toxicity in HMEECs. Cell viability was analyzed using the cell counting kit, and reactive oxygen species (ROS activity was measured using a cellular ROS detection kit. After lead(II acetate trihydrate treatment, mRNA levels of various genes were assessed by semiquantitative real-time polymerase chain reaction. Following treatment with AME or CME after lead exposure, the changes in cell viability, ROS activity, and gene expression were analyzed. Exposure to >100 μg/mL of lead(II acetate trihydrate caused a significant decrease in cell viability and increased ROS production in HMEECs. Lead exposure significantly increased the mRNA expression of genes encoding inflammatory cytokines and mucins. Administration of AME or CME restored cell viability, reduced ROS activity, and ameliorated mRNA levels. Our findings suggest that environmental lead exposure is related to the development of otitis media, and AME and CME may have antioxidative and anti-inflammatory effects against lead toxicity.

  16. Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells.

    Science.gov (United States)

    Wilmer, Martijn J G; de Graaf-Hess, Adriana; Blom, Henk J; Dijkman, Henry B P M; Monnens, Leo A; van den Heuvel, Lambertus P; Levtchenko, Elena N

    2005-11-18

    Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.

  17. Ionizing radiation induces heritable disruption of epithelial cell interactions

    Science.gov (United States)

    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.

  18. Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract.

    Science.gov (United States)

    Li, W C; Spector, A

    1996-01-01

    Epidemiological and experimental studies have revealed that exposure to UV can induce cataractogenesis. To investigate the mechanism of this induction, viability of the lens epithelial cells from UVB-treated rat lenses were examined. Irradiation of the cultured rat lenses with 8 J/s/m2 UVB for 60 min triggers lens epithelial cell apoptosis as determined by terminal deoxyribonucleotide transferase (TdT) labeling and DNA fragmentation assays. The apoptotic lens epithelial cells were initially found in the equatorial region and then quickly appeared in both equatorial and central regions. The percentage of apoptotic cells continuously increased during the postirradiation incubation. After a 5-h post-UVB incubation, more than 50% of the lens epithelial cells were apoptotic. By 24 h, all of the lens epithelial cells in the irradiated lenses were dead through apoptosis. Associated with this apoptotic process is a large upregulation of the proto-oncogene, c-fos. Opacification appears to follow the death of lens epithelial cells occurring first in the equatorial region and then in the central area. This is also true of classical cataract parameters such as non-protein thiol and wet weight, which are significantly modified only after appreciable epithelial cell apoptosis. Together, these results suggest that the rapid apoptotic death of the lens epithelial cells induced by UVB initiates cataract development.

  19. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    NARCIS (Netherlands)

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; de Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing

  20. Physiology and pathophysiology of apoptosis in epithelial cells of the liver, pancreas, and intestine.

    Science.gov (United States)

    Jones, B A; Gores, G J

    1997-12-01

    Cell death of gastrointestinal epithelial cells occurs by a process referred to as apoptosis. In this review, we succinctly define apoptosis and summarize the role of apoptosis in the physiology and pathophysiology of epithelial cells in the liver, pancreas, and small and large intestine. The physiological mediators regulating apoptosis in gastrointestinal epithelial cells, when known, are discussed. Selected pathophysiological consequences of excessive apoptosis and inhibition of apoptosis are used to illustrate the significance of apoptosis in disease processes. These examples demonstrate that excessive apoptosis may result in epithelial cell atrophy, injury, and dysfunction, whereas inhibition of apoptosis results in hyperplasia and promotes malignant transformation. The specific cellular mechanisms responsible for dysregulation of epithelial cell apoptosis during pathophysiological disturbances are emphasized. Potential future areas of physiological research regarding apoptosis in gastrointestinal epithelia are highlighted when appropriate.

  1. Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

    Science.gov (United States)

    Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

    2009-04-01

    Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

  2. Cell cycle control by components of cell anchorage

    OpenAIRE

    Gad, Annica

    2005-01-01

    Extracellular factors, such as growth factors and cell anchorage to the extracellular matrix, control when and where cells may proliferate. This control is abolished when a normal cell transforms into a tumour cell. The control of cell proliferation by cell anchorage was elusive and less well studied than the control by growth factors. Therefore, we aimed to clarify at what points in the cell cycle and through which molecular mechanisms cell anchorage controls cell cycle pro...

  3. Polystyrene nanoparticles activate ion transport in human airway epithelial cells

    Directory of Open Access Journals (Sweden)

    McCarthy J

    2011-06-01

    Full Text Available J McCarthy1, X Gong2, D Nahirney2, M Duszyk2, MW Radomski11School of Pharmacy and Pharmaceutical Sciences, Panoz Institute, Trinity College Dublin, Dublin, Ireland; 2Department of Physiology, University of Alberta, Edmonton, Alberta, CanadaBackground: Over the last decade, nanotechnology has provided researchers with new nanometer materials, such as nanoparticles, which have the potential to provide new therapies for many lung diseases. In this study, we investigated the acute effects of polystyrene nanoparticles on epithelial ion channel function.Methods: Human submucosal Calu-3 cells that express cystic fibrosis transmembrane conductance regulator (CFTR and baby hamster kidney cells engineered to express the wild-type CFTR gene were used to investigate the actions of negatively charged 20 nm polystyrene nanoparticles on short-circuit current in Calu-3 cells by Ussing chamber and single CFTR Cl- channels alone and in the presence of known CFTR channel activators by using baby hamster kidney cell patches.Results: Polystyrene nanoparticles caused sustained, repeatable, and concentration-dependent increases in short-circuit current. In turn, these short-circuit current responses were found to be biphasic in nature, ie, an initial peak followed by a plateau. EC50 values for peak and plateau short-circuit current responses were 1457 and 315.5 ng/mL, respectively. Short-circuit current was inhibited by diphenylamine-2-carboxylate, a CFTR Cl- channel blocker. Polystyrene nanoparticles activated basolateral K+ channels and affected Cl- and HCO3- secretion. The mechanism of short-circuit current activation by polystyrene nanoparticles was found to be largely dependent on calcium-dependent and cyclic nucleotide-dependent phosphorylation of CFTR Cl- channels. Recordings from isolated inside-out patches using baby hamster kidney cells confirmed the direct activation of CFTR Cl- channels by the nanoparticles.Conclusion: This is the first study to identify

  4. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  5. Endothelial induced EMT in breast epithelial cells with stem cell properties.

    Directory of Open Access Journals (Sweden)

    Valgardur Sigurdsson

    Full Text Available Epithelial to mesenchymal transition (EMT is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad to N-Cadherin (N-Cad and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high/CD24(low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

  6. Endothelial induced EMT in breast epithelial cells with stem cell properties.

    Science.gov (United States)

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

    2011-01-01

    Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

  7. Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

    Science.gov (United States)

    Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

    2002-10-01

    To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

  8. Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

    Science.gov (United States)

    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

  9. The genetic and molecular basis of bacterial invasion of epithelial cells

    African Journals Online (AJOL)

    Invasion of epithelial cells was demonstrated to be triggered by invasion plasmid antigens B, C, and D ( IpaB, IpaC and IpaD ) which is accomplished by intracellular spread gene icsA. The invasion of epithelial cells by some individual species of bacteria were also reviewed.Yersinia enterocolitica invasiveness was shown ...

  10. PKC activation induces inflammatory response and cell death in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hyunhee Kim

    Full Text Available A variety of airborne pathogens can induce inflammatory responses in airway epithelial cells, which is a crucial component of host defence. However, excessive inflammatory responses and chronic inflammation also contribute to different diseases of the respiratory system. We hypothesized that the activation of protein kinase C (PKC is one of the essential mechanisms of inflammatory response in airway epithelial cells. In the present study, we stimulated human bronchial lung epithelial (BEAS-2B cells with the phorbol ester Phorbol 12, 13-dibutyrate (PDBu, and examined gene expression profile using microarrays. Microarray analysis suggests that PKC activation induced dramatic changes in gene expression related to multiple cellular functions. The top two interaction networks generated from these changes were centered on NFκB and TNF-α, which are two commonly known pathways for cell death and inflammation. Subsequent tests confirmed the decrease in cell viability and an increase in the production of various cytokines. Interestingly, each of the increased cytokines was differentially regulated at mRNA and/or protein levels by different sub-classes of PKC isozymes. We conclude that pathological cell death and cytokine production in airway epithelial cells in various situations may be mediated through PKC related signaling pathways. These findings suggest that PKCs can be new targets for treatment of lung diseases.

  11. Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

    2009-01-01

    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/β-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/β-catenin signaling were determined, suggested down-regulation of Wnt/β-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3α can inhibit the epithelial differentiation of MSCs. A loss of β-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated β-catenin expression and subsequently decreased β-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/β-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

  12. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    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/.

  13. Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells.

    Science.gov (United States)

    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. Copyright © 2016 the American Physiological Society.

  14. Advanced three-dimensional culture of equine intestinal epithelial stem cells.

    Science.gov (United States)

    Stewart, A Stieler; Freund, J M; Gonzalez, L M

    2018-03-01

    Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Descriptive study. Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease

  15. Analysis of cell-cycle regulation following exposure of lung-derived cells to γ-rays

    Science.gov (United States)

    Trani, D.; Lucchetti, C.; Cassone, M.; D'Agostino, L.; Caputi, M.; Giordano, A.

    Acute exposure of mammalian cells to ionizing radiation results in a delay of cell-cycle progression and/or augmentation of apoptosis. Following ionizing radiation-induced DNA damage, cell-cycle arrest in the G1- or G2-phase of the cell-cycle prevents or delays DNA replication or mitosis, providing time for the DNA repair machinery to exert its function. Deregulation or failing of cell-cycle checkpoints and/or DNA repair mechanisms may lead normal cells bearing chromosome mutations to acquire neoplastic autonomy, which in turn can trigger the onset of cancer. Existing studies have focused on the impact of p53 status on the radiation response of lung cancer (LC) cell lines in terms of both cell-cycle regulation and apoptosis, while no comparative studies have been performed on the radiation response of lung derived normal and cancerous epithelial cells. To investigate the radiation response in normal and cancerous phenotypes, along with the role and impact of p53 status, and possible correlations with pRb/p105 or other proteins involved in carcinogenesis and cell-cycle regulation, we selected two lung-derived epithelial cell lines, one normal (NL20, p53 wild-type) and one non-small cell lung cancer (NSCLC), H358 (known to be p53-deficient). We compared the levels of γ-induced cell proliferation ability, cell-cycle arrest, apoptotic index, and expression levels of cell-cycle regulating and regulated proteins. The different cell sensitivity, apoptotic response and protein expression profiles resulting from our study for NL20 and H358 cells suggest that still unknown mechanisms involving p53, pRb/p105 and their target molecules might play a pivotal role in determining cell sensitivity and resistance upon exposure to ionizing radiation.

  16. Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

    Directory of Open Access Journals (Sweden)

    Bhavani S. Kowtharapu

    2018-02-01

    Full Text Available Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM on corneal epithelial cell function along with substance P (SP. Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK, paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained

  17. Collective cell streams in epithelial monolayers depend on cell adhesion

    International Nuclear Information System (INIS)

    Czirók, András; Varga, Katalin; Méhes, Előd; Szabó, András

    2013-01-01

    We report spontaneously emerging, randomly oriented, collective streaming behavior within a monolayer culture of a human keratinocyte cell line, and explore the effect of modulating cell adhesions by perturbing the function of calcium-dependent cell adhesion molecules. We demonstrate that decreasing cell adhesion induces narrower and more anisotropic cell streams, reminiscent of decreasing the Taylor scale of turbulent liquids. To explain our empirical findings, we propose a cell-based model that represents the dual nature of cell–cell adhesions. Spring-like connections provide mechanical stability, while a cellular Potts model formalism represents surface-tension driven attachment. By changing the relevance and persistence of mechanical links between cells, we are able to explain the experimentally observed changes in emergent flow patterns. (paper)

  18. Gene expression in epithelial cells in response to pneumovirus infection

    Directory of Open Access Journals (Sweden)

    Rosenberg Helene F

    2001-05-01

    Full Text Available Abstract Respiratory syncytial virus (RSV and pneumonia virus of mice (PVM are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner.

  19. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob

    2003-01-01

    Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers ...... of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium.......Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers...

  20. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  1. Characterization of kidney epithelial cells from the Florida manatee, Trichechus manatus latirostris.

    Science.gov (United States)

    Sweat JMDunigan, D D; Wright, S D

    2001-06-01

    The West-Indian manatee, Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5 x 10(3) cells/cm2, and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.

  2. Small-Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Alexandra Mikhailova

    2014-02-01

    Full Text Available Human induced pluripotent stem cells (hiPSCs offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation.

  3. Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

    Science.gov (United States)

    Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

    2015-08-01

    Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Allergen recognition by innate immune cells: critical role of dendritic and epithelial cells

    Directory of Open Access Journals (Sweden)

    Fabian eSalazar

    2013-11-01

    Full Text Available Allergy is an exacerbated response of the immune system against non-self-proteins called allergens and is typically characterized by biased type-2 T helper cell and deleterious IgE mediated immune responses. The allergic cascade starts with the recognition of allergens by antigen presenting cells, mainly dendritic cells, culminating in mast cell sensitization and triggering. Dendritic cells have been demonstrated to play a crucial role in orchestrating allergic diseases. Using different C-type lectin receptors dendritic cells are able to recognize and internalize a number of allergens from diverse sources leading to sensitization. Furthermore, there is increasing evidence highlighting the role of epithelial cells in triggering and modulating immune responses to allergens. As well as providing a physical barrier, epithelial cells can interact with allergens and influence dendritic cells behaviour through the release of a number of Th2 promoting cytokines. In this review we will summarise current understanding of how allergens are recognised by dendritic cells and epithelial cells and what are the consequences of such interaction in the context of allergic sensitisation and downstream events leading to allergic inflammation. Better understanding of the molecular mechanisms of allergen recognition and associated signalling pathways could enable developing more effective therapeutic strategies that target the initial steps of allergic sensitisation hence hindering development or progression of allergic diseases.

  5. File list: ALL.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. Interferon lambda inhibits dengue virus replication in epithelial cells.

    Science.gov (United States)

    Palma-Ocampo, Helen K; Flores-Alonso, Juan C; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Flores-Mendoza, Lilian; Herrera-Camacho, Irma; Rosas-Murrieta, Nora H; Santos-López, Gerardo

    2015-09-28

    In viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection. Dengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR. We found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG's peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression. Evidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

  9. Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

    Science.gov (United States)

    Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

    2017-08-15

    Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

  10. Epithelial cells as active player in fibrosis: findings from an in vitro model.

    Directory of Open Access Journals (Sweden)

    Solange Moll

    Full Text Available Kidney fibrosis, a scarring of the tubulo-interstitial space, is due to activation of interstitial myofibroblasts recruited locally or systemically with consecutive extracellular matrix deposition. Newly published clinical studies correlating acute kidney injury (AKI to chronic kidney disease (CKD challenge this pathological concept putting tubular epithelial cells into the spotlight. In this work we investigated the role of epithelial cells in fibrosis using a simple controlled in vitro system. An epithelial/mesenchymal 3D cell culture model composed of human proximal renal tubular cells and fibroblasts was challenged with toxic doses of Cisplatin, thus injuring epithelial cells. RT-PCR for classical fibrotic markers was performed on fibroblasts to assess their modulation toward an activated myofibroblast phenotype in presence or absence of that stimulus. Epithelial cell lesion triggered a phenotypical modulation of fibroblasts toward activated myofibroblasts as assessed by main fibrotic marker analysis. Uninjured 3D cell culture as well as fibroblasts alone treated with toxic stimulus in the absence of epithelial cells were used as control. Our results, with the caveats due to the limited, but highly controllable and reproducible in vitro approach, suggest that epithelial cells can control and regulate fibroblast phenotype. Therefore they emerge as relevant target cells for the development of new preventive anti-fibrotic therapeutic approaches.

  11. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

    Directory of Open Access Journals (Sweden)

    María Laura Chiribao

    2014-01-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response, a great number of transcription factors (including the majority of NFκB family members, and host metabolism (cholesterol, fatty acids, and phospholipids. These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

  12. Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

    Science.gov (United States)

    Ziauddin, S M; Yoshimura, A; Montenegro Raudales, J L; Ozaki, Y; Higuchi, K; Ukai, T; Kaneko, T; Miyazaki, T; Latz, E; Hara, Y

    2018-06-01

    Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1β precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental

  13. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells

    International Nuclear Information System (INIS)

    Sides, Mark D.; Block, Gregory J.; Shan, Bin; Esteves, Kyle C.; Lin, Zhen; Flemington, Erik K.; Lasky, Joseph A.

    2011-01-01

    Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies.

  14. Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells.

    Science.gov (United States)

    Payne, Emily H; Ramalingam, Dhivya; Fox, Donald T; Klotman, Mary E

    2018-01-15

    Prior studies have found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy have remained unclear. Here we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). We found that as in many cell types, Vpr first initiates G 2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G 2 -arrested RTECs reenter the cell cycle. Following this cell cycle reentry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small-molecule inhibitors of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G 2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G 2 arrest to polyploidy. These findings outline a temporal, molecularly regulated path to polyploidy in HIV-positive renal cells. IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long-term persistence of HIV in compartments. The kidney is recognized as one such compartment, since viral DNA and mRNA persist in the renal tissues of HIV-positive patients. Further, renal disease is a long-term comorbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish

  15. GATA4 Regulates Epithelial Cell Proliferation to Control Intestinal Growth and Development in MiceSummary

    Directory of Open Access Journals (Sweden)

    Bridget M. Kohlnhofer

    2016-03-01

    Full Text Available Background & Aims: The embryonic small intestinal epithelium is highly proliferative, and although much is known about mechanisms regulating proliferation in the adult intestine, the mechanisms controlling epithelial cell proliferation in the developing intestine are less clear. GATA4, a transcription factor that regulates proliferation in other developing tissues, is first expressed early in the developing gut in midgut endoderm. GATA4 function within midgut endoderm and the early intestinal epithelium is unknown. Methods: By using Sonic Hedgehog Cre to eliminate GATA4 in the midgut endoderm of mouse embryos, we determined the impact of loss of GATA4 on intestinal development, including epithelial cell proliferation, between embryonic day (E9.5 and E18.5. Results: We found that intestinal length and width were decreased in GATA4 mutants compared with controls. GATA4-deficient intestinal epithelium contained fewer cells, and epithelial girth was decreased. We further observed a decreased proportion of proliferating epithelial cells at E10.5 and E11.5 in GATA4 mutants. We showed that GATA4 binds to chromatin containing GATA4 consensus binding sites within cyclin D2 (Ccnd2, cyclin-dependent kinase 6 (Cdk6, and frizzled 5 (Fzd5. Moreover, Ccnd2, Cdk6, and Fzd5 transcripts were reduced at E11.5 in GATA4 mutant tissue. Villus morphogenesis was delayed, and villus structure was abnormal in GATA4 mutant intestine. Conclusions: Our data identify GATA4 as an essential regulator of early intestinal epithelial cell proliferation. We propose that GATA4 controls proliferation in part by directly regulating transcription of cell-cycle mediators. Our data further suggest that GATA4 affects proliferation through transcriptional regulation of Fzd5, perhaps by influencing the response of the epithelium to WNT signaling. Keywords: Transcriptional Regulation, WNT Signaling, Villus Morphogenesis

  16. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

    Directory of Open Access Journals (Sweden)

    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  17. A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

    Science.gov (United States)

    Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

    2012-01-01

    In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

  18. The Regenerative Potential of Parietal Epithelial Cells in Adult Mice

    Science.gov (United States)

    Berger, Katja; Schulte, Kevin; Boor, Peter; Kuppe, Christoph; van Kuppevelt, Toin H.; Floege, Jürgen; Smeets, Bart

    2014-01-01

    Previously, we showed that some podocytes in juvenile mice are recruited from cells lining Bowman’s capsule, suggesting that parietal epithelial cells (PECs) are a progenitor cell population for podocytes. To investigate whether PECs also replenish podocytes in adult mice, PECs were genetically labeled in an irreversible fashion in 5-week-old mice. No significant increase in labeled podocytes was observed, even after 18 months. To accelerate a potential regenerative mechanism, progressive glomerular hypertrophy was induced by progressive partial nephrectomies. Again, no significant podocyte replenishment was observed. Rather, labeled PECs exclusively invaded segments of the tuft affected by glomerulosclerosis, consistent with our previous findings. We next reassessed PEC recruitment in juvenile mice using a different reporter mouse and confirmed significant recruitment of labeled PECs onto the glomerular tuft. Moreover, some labeled cells on Bowman’s capsule expressed podocyte markers, and cells on Bowman’s capsule were also directly labeled in juvenile podocyte-specific Pod-rtTA transgenic mice. In 6-week-old mice, however, cells on Bowman’s capsule no longer expressed podocyte-specific markers. Similarly, in human kidneys, some cells on Bowman’s capsule expressed the podocyte marker synaptopodin from 2 weeks to 2 years of age but not at 7 years of age. In summary, podocyte regeneration from PECs could not be detected in aging mice or models of glomerular hypertrophy. We propose that a small fraction of committed podocytes reside on Bowman’s capsule close to the vascular stalk and are recruited onto the glomerular tuft during infancy to adolescence in mice and humans. PMID:24408873

  19. Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection

    DEFF Research Database (Denmark)

    Holck, Susanne; Holm, I.L.; Holck, P.P.

    2007-01-01

    Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytoki...

  20. Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Mytych, Jennifer, E-mail: jennifermytych@gmail.com [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland)

    2017-01-15

    Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

  1. Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

    International Nuclear Information System (INIS)

    Mytych, Jennifer; Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek

    2017-01-01

    Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

  2. Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in Rhesus monkeys exposed to ozone

    International Nuclear Information System (INIS)

    Castleman, W.L.; Dungworth, D.L.; Schwartz, L.W.; Tyler, W.S.

    1980-01-01

    The pathogenesis of acute respiratory bronchiolitis was examined in Rhesus monkeys exposed to 0.8 ppM ozone for 4 to 50 hours. Epithelial injury and renewal were qualitatively and quantitatively characterized by correlated techniques of scanning and transmission electron microscopy as well as by light-microscopic autoradiography following labeling with tritiated thymidine. Extensive degeneration and necrosis of Type 1 epithelial cells occurred on the respiratory bronchiolar wall during the initial 4 to 12 hours of exposure. Increased numbers of labeled epithelial cells were present in this region after 18 hours of exposure, and the highest labeling index (18%) was measured after 50 hours of exposure. Most (67 to 80%) of the labeled cells and all the mitotic epithelial cells (22) observed ultrastructurally were cuboidal bronchiolar epithelial cells. Of the labeled epithelial cells, 20 to 33% were Type 2 epithelial cells. After 50 hours of exposure the respiratory bronchiolar epithelium was hyperplastic. The predominant inflammatory cell in respiratory bronchiolar exudate was the alveolar macrophage. Monkeys that were exposed for 50 hours and allowed to recover in unozonized air for 7 days had incomplete resolution of respiratory bronchiolar epithelial hyperplasia. The results indicate that Type 1 epithelial cells lining respiratory bronchioles are the cell types most sensitive to injury and that both cuboidal bronchiolar epithelial cells and Type 2 epithelial cells function as stem cells in epithelial renewal

  3. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    Directory of Open Access Journals (Sweden)

    Z. Liu

    2010-05-01

    Full Text Available Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines, allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39 and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV, may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

  4. MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

    Science.gov (United States)

    Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

    2018-02-02

    Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

  5. Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

    OpenAIRE

    Foltz, LP; Clegg, DO

    2017-01-01

    We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embry...

  6. Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

    Science.gov (United States)

    Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

    2012-01-01

    Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

  7. Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Millaku, Agron, E-mail: agron.mi@hotmail.com [Limnos-Company for Applied Ecology Ltd, Podlimbarskega 31, 1000 Ljubljana (Slovenia); Drobne, Damjana [University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana (Slovenia); Centre of Excellence, Advanced Materials and Technologies for the Future (CO NAMASTE), Jamova cesta 39, 1000 Ljubljana (Slovenia); Centre of Excellence, Nanoscience and Nanotechnology (Nanocentre), Jamova cesta 39, 1000 Ljubljana (Slovenia); Torkar, Matjaz [Institute of Metals and Technology IMT, Lepi pot 11, 1000 Ljubljana (Slovenia); Jožef Stefan Institute, Condensed Matter Physics Department, Jamova cesta 39, 1000 Ljubljana (Slovenia); Novak, Sara [University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana (Slovenia); Remškar, Maja [Jožef Stefan Institute, Condensed Matter Physics Department, Jamova cesta 39, 1000 Ljubljana (Slovenia); Pipan-Tkalec, Živa [University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana (Slovenia)

    2013-09-15

    Graphical abstract: Scanning electron microscopy is particularly well suited to the observation of nanofibre/cell interaction in the endothelial cells lining the hepatopancreas. (a) Tungsten oxide nanofibres, (b) test organism Porcellio scaber and schematic appearance of digestive tubes, (c) digestive tube (hepatopancreas) prepared for SEM investigation, (d) digestive gland cells (C) with nanofibres (NF) embedded in the cell membrane and (e) nanofibres inserted deeply in the cells and damaged nanofibres due to peristalsis. -- Highlights: • Tungsten oxide nanofibres react physically with digestive gland epithelial cells in Porcellio scaber. • Physical peristaltic forces of lead to insertion of nanofibres into the cells. • No toxic responses as measured by conventional toxicity biomarkers were detected. • Physical interactions were observed in a majority of the investigated animals. -- Abstract: 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.

  8. Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells

    International Nuclear Information System (INIS)

    Millaku, Agron; Drobne, Damjana; Torkar, Matjaz; Novak, Sara; Remškar, Maja; Pipan-Tkalec, Živa

    2013-01-01

    Graphical abstract: Scanning electron microscopy is particularly well suited to the observation of nanofibre/cell interaction in the endothelial cells lining the hepatopancreas. (a) Tungsten oxide nanofibres, (b) test organism Porcellio scaber and schematic appearance of digestive tubes, (c) digestive tube (hepatopancreas) prepared for SEM investigation, (d) digestive gland cells (C) with nanofibres (NF) embedded in the cell membrane and (e) nanofibres inserted deeply in the cells and damaged nanofibres due to peristalsis. -- Highlights: • Tungsten oxide nanofibres react physically with digestive gland epithelial cells in Porcellio scaber. • Physical peristaltic forces of lead to insertion of nanofibres into the cells. • No toxic responses as measured by conventional toxicity biomarkers were detected. • Physical interactions were observed in a majority of the investigated animals. -- Abstract: 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. Protein tyrosine nitration in the cell cycle

    International Nuclear Information System (INIS)

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-01-01

    Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  10. Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jason Bennett

    2016-04-01

    Full Text Available Epithelial-mesenchymal transition (EMT, a process which describes the trans-differentiation of epithelial cells into motile mesenchymal cells, is pivotal in stem cell behavior, development and wound healing, as well as contributing to disease processes including fibrosis and cancer progression. Maintenance immunosuppression with calcineurin inhibitors (CNIs has become routine management for renal transplant patient, but unfortunately the nephrotoxicity of these drugs has been well documented. HK-2 cells were exposed to Tacrolimus (FK506 and EMT markers were assessed by RT PCR and western blot. FK506 effects on TGF-β mRNA were assessed by RT PCR and TGF-β secretion was measured by ELISA. The impact of increased TGF-β secretion on Smad signaling pathways was investigated. The impact of inhibition of TGF-β signaling on EMT processes was assessed by scratch-wound assay. The results presented in this study suggest that FK506 initiates EMT processes in the HK-2 cell line, with altered expression of epithelial and myofibroblast markers evident. Additionally, the study demonstrates that FK506 activation of the TGF-β/ SMAD pathways is an essential step in the EMT process. Overall the results demonstrate that EMT is heavily involved in renal fibrosis associated with CNI nephrotoxicity.

  11. Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro.

    Science.gov (United States)

    Clouzeau, Chloé; Godefroy, David; Riancho, Luisa; Rostène, William; Baudouin, Christophe; Brignole-Baudouin, Françoise

    2012-01-01

    Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays. The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells. As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from

  12. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

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

  13. Feature Importance for Human Epithelial (HEp-2 Cell Image Classification

    Directory of Open Access Journals (Sweden)

    Vibha Gupta

    2018-02-01

    Full Text Available Indirect Immuno-Fluorescence (IIF microscopy imaging of human epithelial (HEp-2 cells is a popular method for diagnosing autoimmune diseases. Considering large data volumes, computer-aided diagnosis (CAD systems, based on image-based classification, can help in terms of time, effort, and reliability of diagnosis. Such approaches are based on extracting some representative features from the images. This work explores the selection of the most distinctive features for HEp-2 cell images using various feature selection (FS methods. Considering that there is no single universally optimal feature selection technique, we also propose hybridization of one class of FS methods (filter methods. Furthermore, the notion of variable importance for ranking features, provided by another type of approaches (embedded methods such as Random forest, Random uniform forest is exploited to select a good subset of features from a large set, such that addition of new features does not increase classification accuracy. In this work, we have also, with great consideration, designed class-specific features to capture morphological visual traits of the cell patterns. We perform various experiments and discussions to demonstrate the effectiveness of FS methods along with proposed and a standard feature set. We achieve state-of-the-art performance even with small number of features, obtained after the feature selection.

  14. Radiosensitizing effect of epothilone B on human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Baumgart, T.; Kriesen, S.; Hildebrandt, G.; Manda, K. [Univ. of Rostock (Germany). Dept. of Radiotherapy and Radiation Oncology; Klautke, G.; Fietkau, R. [Friedrich-Alexander-Univ. Erlangen-Nuernberg, Erlangen (Germany). Dept. of Radiation Oncology; Kuznetsov, S.A.; Weiss, D.G. [Univ. of Rostock (Germany). Inst. of Biological Sciences, Cell Biology, and Biosystems Technology

    2012-02-15

    A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly. Cancer cells were treated with epothilone B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a {gamma}H2AX foci assay was used. To determine the effect of epothilone B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted. Epothilone B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC{sub 50} values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone B. The {gamma}H2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin. The results of this study showed that epothilone B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone B. It was also demonstrated that epothilone B in fact targets microtubules in a more effective manner than paclitaxel. (orig.)

  15. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

    Directory of Open Access Journals (Sweden)

    Ludmila Alekseeva

    Full Text Available Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.

  16. Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation.

    Science.gov (United States)

    Ali, Niwa; Zirak, Bahar; Rodriguez, Robert Sanchez; Pauli, Mariela L; Truong, Hong-An; Lai, Kevin; Ahn, Richard; Corbin, Kaitlin; Lowe, Margaret M; Scharschmidt, Tiffany C; Taravati, Keyon; Tan, Madeleine R; Ricardo-Gonzalez, Roberto R; Nosbaum, Audrey; Bertolini, Marta; Liao, Wilson; Nestle, Frank O; Paus, Ralf; Cotsarelis, George; Abbas, Abul K; Rosenblum, Michael D

    2017-06-01

    The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of T regs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

    Science.gov (United States)

    Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

    2013-09-24

    The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  17. Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

    International Nuclear Information System (INIS)

    Shin, Jung Ar; Chung, Jin Sil; Cho, Sang-Ho; Kim, Hyung Jung; Yoo, Young Do

    2013-01-01

    Highlights: •Romo1 mediates oxidative stress-induced mitochondrial ROS production. •Romo1 induction by oxidative stress plays an important role in oxidative stress-induced apoptosis. •Romo1 overexpression correlates with epithelial cell death in patients with IPF. -- Abstract: Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H 2 O 2 ) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H 2 O 2 treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-15

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

  19. Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jung Ar [Department of Internal Medicine, Yonsei University College of Medicine, Yonsei University Health System, Seoul 135-270 (Korea, Republic of); Chung, Jin Sil [Laboratory of Molecular Cell Biology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Cho, Sang-Ho [Department of Pathology, Pochon CHA University, College of Medicine, Gyeonggi-do (Korea, Republic of); Kim, Hyung Jung, E-mail: khj57@yuhs.ac.kr [Department of Internal Medicine, Yonsei University College of Medicine, Yonsei University Health System, Seoul 135-270 (Korea, Republic of); Yoo, Young Do, E-mail: ydy1130@korea.ac.kr [Laboratory of Molecular Cell Biology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of)

    2013-09-20

    Highlights: •Romo1 mediates oxidative stress-induced mitochondrial ROS production. •Romo1 induction by oxidative stress plays an important role in oxidative stress-induced apoptosis. •Romo1 overexpression correlates with epithelial cell death in patients with IPF. -- Abstract: Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H{sub 2}O{sub 2}) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H{sub 2}O{sub 2} treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.

  20. Contraction and elongation: Mechanics underlying cell boundary deformations in epithelial tissue.

    Science.gov (United States)

    Hara, Yusuke

    2017-06-01

    The cell-cell boundaries of epithelial cells form cellular frameworks at the apical side of tissues. Deformations in these boundaries, for example, boundary contraction and elongation, and the associated forces form the mechanical basis of epithelial tissue morphogenesis. In this review, using data from recent Drosophila studies on cell boundary contraction and elongation, I provide an overview of the mechanism underlying the bi-directional deformations in the epithelial cell boundary, that are sustained by biased accumulations of junctional and apico-medial non-muscle myosin II. Moreover, how the junctional tensions exist on cell boundaries in different boundary dynamics and morphologies are discussed. Finally, some future perspectives on how recent knowledge about single cell boundary-level mechanics will contribute to our understanding of epithelial tissue morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

  1. Altered expression of epithelial cell surface glycoconjugates and intermediate filaments at the margins of mucosal wounds

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Grøn, B.; Mandel, U.

    1998-01-01

    Alterations in cell to cell adhesion are necessary to enable the type of cell movements that are associated with epithelial wound healing and malignant invasion. Several studies of transformed cells have related epithelial cell movement to changes in the cell surface expression of the carbohydrate......-T antigen. The changes induced by wounding in the expression of collagen IV, laminin gamma2-chain (laminin-5), and laminin alpha5-chain were similar to those found in skin wounds and served to define the region of epithelial movement. This region was found to show a marked increase in staining for both...... epithelium, a pattern of expression similar to K16, which was also strongly upregulated in both the outgrowth and the adjacent nonwounded epithelium. These findings provide further support for an influence of such carbohydrate structures on the migratory behavior of epithelial cells....

  2. Transcriptional Waves in the Yeast Cell Cycle

    OpenAIRE

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-01-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillat...

  3. Epithelial cells in nipple aspirate fluid and subsequent breast cancer risk: A historic prospective study

    International Nuclear Information System (INIS)

    Baltzell, Kimberly A; Moghadassi, Michelle; Rice, Terri; Sison, Jennette D; Wrensch, Margaret

    2008-01-01

    Past studies have shown that women with abnormal cytology or epithelial cells in nipple aspirate fluid (NAF) have an increased relative risk (RR) of breast cancer when compared to women from whom NAF was attempted but not obtained (non-yielders). This study analyzed NAF results from a group of women seen in a breast clinic between 1970–1991 (N = 2480). Our analysis presented here is an aggregate of two sub-groups: women with questionnaire data (n = 712) and those with NAF visits beginning in 1988 (n = 238), the year in which cancer case information was uniformly collected in California. Cytological classification was determined for a group of 946 women using the most abnormal epithelial cytology observed in fluid specimens. Breast cancer incidence and mortality status was determined through June 2006 using data from the California Cancer Registry, California Vital Statistics and self-report. We estimated odd ratios (ORs) for breast cancer using logistic regression analysis, adjusting for age. We analyzed breast cancer risk related to abnormality of NAF cytology using non-yielders as the referent group and breast cancer risk related to the presence or absence of epithelial cells in NAF, using non-yielders/fluid without epithelial cells as the referent group. Overall, 10% (93) of the 946 women developed breast cancer during the follow-up period. Age-adjusted ORs and 95% confidence intervals (C.I.) compared to non-yielders were 1.4 (0.3 to 6.4), 1.7 (0.9 to 3.5), and 2.0 (1.1 to 3.6) for women with fluid without epithelial cells, normal epithelial cells and hyperplasia/atypia, respectively. Comparing the presence or absence of epithelial cells in NAF, women with epithelial cells present in NAF were more likely to develop breast cancer than non-yielders or women with fluid without epithelial cells (RR = 1.9, 1.2 to 3.1). These results support previous findings that 1) women with abnormal epithelial cells in NAF have an increased risk of breast cancer when compared to

  4. Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells

    NARCIS (Netherlands)

    van Schilfgaarde, M.; van Alphen, L.; Eijk, P.; Everts, V.; Dankert, J.

    1995-01-01

    Haemophilus influenzae penetrates the respiratory epithelium during carriage and invasive disease, including respiratory tract infections. We developed an in vitro model system consisting of lung epithelial NCI-H292 cells on permeable supports to study the passage of H. influenzae through lung

  5. Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

    Directory of Open Access Journals (Sweden)

    Jennifer S. Liu

    2012-11-01

    Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

  6. Regulation of cell cycle progression by cell-cell and cell-matrix forces

    NARCIS (Netherlands)

    Uroz, Marina; Wistorf, Sabrina; Serra-Picamal, Xavier; Conte, Vito; Sales-Pardo, Marta; Roca-Cusachs, Pere; Guimerà, Roger; Trepat, Xavier

    2018-01-01

    It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces 1-12 . However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression

  7. Action of cholera toxin in the intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with the cell membrane. This involves a large number (17 million per cell) of high affinity binding sites which belong to a single class. Binding of biologically active 125 I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected in the isolated cells. The response (elevation of cellular cAMP) of the enterocytes to cholera toxin is linear with time for 40-50 min and causes a six- to eight-fold increase over control levels at steady stae. cAMP and agents that increase cAMP production inhibit Cl - -independent Na + influx into the isolated enterocytes whereas chlorporomazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na + entry. Correlation between cellular cAMP levels and the magnitude of Na + influx into the enterocytes provides evidence for a cAMP-mediated control of intestinal Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT and Na + during induction of intestinal secretion. The effect of cAMP on Na + but no Cl - influx in our villus cell preparation can be partially explained in terms of a cAMP-regulated Na + /H + neutral exchange system

  8. Proinflammatory effects of cookstove emissions on human bronchial epithelial cells.

    Science.gov (United States)

    Hawley, B; Volckens, J

    2013-02-01

    Approximately half of the world's population uses biomass fuel for indoor cooking and heating. This form of combustion typically occurs in open fires or primitive stoves. Human exposure to emissions from indoor biomass combustion is a global health concern, causing an estimated 1.5 million premature deaths each year. Many 'improved' stoves have been developed to address this concern; however, studies that examine exposure-response with cleaner-burning, more efficient stoves are few. The objective of this research was to evaluate the effects of traditional and cleaner-burning stove emissions on an established model of the bronchial epithelium. We exposed well-differentiated, normal human bronchial epithelial cells to emissions from a single biomass combustion event using either a traditional three-stone fire or one of two energy-efficient stoves. Air-liquid interface cultures were exposed using a novel, aerosol-to-cell deposition system. Cellular expression of a panel of three pro-inflammatory markers was evaluated at 1 and 24 h following exposure. Cells exposed to emissions from the cleaner-burning stoves generated significantly fewer amounts of pro-inflammatory markers than cells exposed to emissions from a traditional three-stone fire. Particulate matter emissions from each cookstove were substantially different, with the three-stone fire producing the largest concentrations of particles (by both number and mass). This study supports emerging evidence that more efficient cookstoves have the potential to reduce respiratory inflammation in settings where solid fuel combustion is used to meet basic domestic needs. Emissions from more efficient, cleaner-burning cookstoves produced less inflammation in well-differentiated bronchial lung cells. The results support evidence that more efficient cookstoves can reduce the health burden associated with exposure to indoor pollution from the combustion of biomass. © 2012 John Wiley & Sons A/S.

  9. Trichomonas vaginalis Contact-Dependent Cytolysis of Epithelial Cells

    Science.gov (United States)

    Lustig, Gila; Ryan, Christopher M.; Secor, W. Evan

    2013-01-01

    Trichomonas vaginalis is an extracellular protozoan parasite that binds to the epithelium of the human urogenital tract during infection. In this study, we examined the propensities of 26 T. vaginalis strains to bind to and lyse prostate (BPH-1) and ectocervical (Ect1) epithelium and to lyse red blood cells (RBCs). We found that only three of the strains had a statistically significant preference for either BPH-1 (MSA1103) or Ect1 (LA1 and MSA1123). Overall, we observed that levels of adherence are highly variable among strains, with a 12-fold range of adherence on Ect1 cells and a 45-fold range on BPH-1 cells. Cytolysis levels displayed even greater variability, from no detectable cytolysis to 80% or 90% cytolysis of Ect1 and BPH-1, respectively. Levels of adherence and cytolysis correlate for weakly adherent/cytolytic strains, and a threshold of attachment was found to be necessary to trigger cytolysis; however, this threshold can be reached without inducing cytolysis. Furthermore, cytolysis was completely blocked when we prevented attachment of the parasites to host cells while allowing soluble factors complete access. We demonstrate that hemolysis was a rare trait, with only 4 of the 26 strains capable of lysing >20% RBCs with a 1:30 parasite/RBC ratio. Hemolysis also did not correlate with adherence to or cytolysis of either male (BPH-1)- or female (Ect1)-derived epithelial cell lines. Our results reveal that despite a broad range of pathogenic properties among different T. vaginalis strains, all strains show strict contact-dependent cytolysis. PMID:23429535

  10. Podocyte and Parietal Epithelial Cell Interactions in Health and Disease.

    Science.gov (United States)

    Al Hussain, Turki; Al Mana, Hadeel; Hussein, Maged H; Akhtar, Mohammed

    2017-01-01

    The glomerulus has 3 resident cells namely mesangial cells that produce the mesangial matrix, endothelial cells that line the glomerular capillaries, and podocytes that cover the outer surface of the glomerular basement membrane. Parietal epithelial cells (PrECs), which line the Bowman's capsule are not part of the glomerular tuft but may have an important role in the normal function of the glomerulus. A significant progress has been made in recent years regarding our understanding of the role and function of these cells in normal kidney and in kidneys with various types of glomerulopathy. In crescentic glomerulonephritis necrotizing injury of the glomerular tuft results in activation and leakage of fibrinogen which provides the trigger for excessive proliferation of PrECs giving rise to glomerular crescents. In cases of collapsing glomerulopathy, podocyte injury causes collapse of the glomerular capillaries and activation and proliferation of PrECs, which accumulate within the urinary space in the form of pseudocrescents. Many of the noninflammatory glomerular lesions such as focal segmental glomerulosclerosis and global glomerulosclerosis also result from podocyte injury which causes variable loss of podocytes. In these cases podocyte injury leads to activation of PrECs that extend on to the glomerular tuft where they cause segmental and/or global sclerosis by producing excess matrix, resulting in obliteration of the capillary lumina. In diabetic nephropathy, in addition to increased matrix production in the mesangium and glomerular basement membranes, increased loss of podocytes is an important determinant of long-term prognosis. Contrary to prior belief there is no convincing evidence for an active podocyte proliferation in any of the above mentioned glomerulopathies.

  11. Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

    Science.gov (United States)

    Bhargava, Maneesh

    Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

  12. Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

    Directory of Open Access Journals (Sweden)

    Guillaume Golovkine

    2016-01-01

    Full Text Available To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.

  13. Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Nir eOsherov

    2012-09-01

    Full Text Available Aspergillus fumigatus is an opportunistic environmental mold that can cause severe allergic responses in atopic individuals and poses a life-threatening risk for severely immunocompromised patients. Infection is caused by inhalation of fungal spores (conidia into the lungs. The initial point of contact between the fungus and the host is a monolayer of lung epithelial cells. Understanding how these cells react to fungal contact is crucial to elucidating the pathobiology of Aspergillus-related disease states. The experimental systems, both in vitro and in vivo, used to study these interactions, are described. Distinction is made between bronchial and alveolar epithelial cells. The experimental findings suggest that lung epithelial cells are more than just innocent bystanders or a purely physical barrier against infection. They can be better described as an active extension of our innate immune system, operating as a surveillance mechanism that can specifically identify fungal spores and activate an offensive response to block infection. This response includes the internalization of adherent conidia and the release of cytokines, antimicrobial peptides and reactive oxygen species. In the case of allergy, lung epithelial cells can dampen an over-reactive immune response by releasing anti-inflammatory compounds such as kinurenine. This review summarizes our current knowledge regarding the interaction of A. fumigatus with lung epithelial cells. A better understanding of the interactions between A. fumigatus and lung epithelial cells has therapeutic implications, as stimulation or inhibition of the epithelial response may alter disease outcome.

  14. Plasticity between Epithelial and Mesenchymal States Unlinks EMT from Metastasis-Enhancing Stem Cell Capacity

    Directory of Open Access Journals (Sweden)

    Evelyne Beerling

    2016-03-01

    Full Text Available Forced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT has been reported to alter metastasis by changing migration and stem cell capacity of tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells may adapt transient and reversible states. Here, we have tested the existence and role of epithelial-mesenchymal plasticity in metastasis of mammary tumors without artificially modifying EMT regulators. In these tumors, we found by intravital microscopy that the motile tumor cells have undergone EMT, while their epithelial counterparts were not migratory. Moreover, we found that epithelial-mesenchymal plasticity renders any EMT-induced stemness differences, as reported previously, irrelevant for metastatic outgrowth, because mesenchymal cells that arrive at secondary sites convert to the epithelial state within one or two divisions, thereby obtaining the same stem cell potential as their arrived epithelial counterparts. We conclude that epithelial-mesenchymal plasticity supports migration but additionally eliminates stemness-enhanced metastatic outgrowth differences.

  15. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    Science.gov (United States)

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  16. Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Dagher Hayat

    2010-02-01

    Full Text Available Abstract Background Transforming growth factor β1 (TGF-β1-mediated epithelial mesenchymal transition (EMT of alveolar epithelial cells (AEC may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ and ciglitazone (CGZ to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker and N-cadherin (mesenchymal cell marker, and collagen 1α1 (COL1A1, CTGF and MMP-2 mRNA. Methods Serum-deprived A549 cells (human AEC cell line were pre-incubated with RGZ and CGZ (1 - 30 μM in the absence or presence of the PPARγ antagonist GW9662 (10 μM before TGFβ-1 (0.075-7.5 ng/ml treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR. Results TGFβ-1 (2.5 ng/ml-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml. However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml, with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ. Conclusions RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPAR

  17. A secreted Salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration

    OpenAIRE

    Lee, Catherine A.; Silva, Milton; Siber, Andrew M.; Kelly, Aaron J.; Galyov, Edouard; McCormick, Beth A.

    2000-01-01

    In response to Salmonella typhimurium, the intestinal epithelium generates an intense inflammatory response consisting largely of polymorphonuclear leukocytes (neutrophils, PMN) migrating toward and ultimately across the epithelial monolayer into the intestinal lumen. It has been shown that bacterial-epithelial cell interactions elicit the production of inflammatory regulators that promote transepithelial PMN migration. Although S. typhimurium can enter intestinal ...

  18. Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth

    DEFF Research Database (Denmark)

    Green, M R; Basketter, D A; Couchman, J R

    1983-01-01

    receptors are detected on the epithelial cells overlying the basement membranes of the epidermis, sebaceous gland, and regions of the hair follicle all of which have proliferative capacity. In marked contrast, tissues which have started to differentiate and lost their growth potential, carry either...... and temporal control of epithelial proliferation....

  19. A Critical Role of TET1/2 Proteins in Cell-Cycle Progression of Trophoblast Stem Cells

    Directory of Open Access Journals (Sweden)

    Stephanie Chrysanthou

    2018-04-01

    Full Text Available Summary: The ten-eleven translocation (TET proteins are well known for their role in maintaining naive pluripotency of embryonic stem cells. Here, we demonstrate that, jointly, TET1 and TET2 also safeguard the self-renewal potential of trophoblast stem cells (TSCs and have partially redundant roles in maintaining the epithelial integrity of TSCs. For the more abundantly expressed TET1, we show that this is achieved by binding to critical epithelial genes, notably E-cadherin, which becomes hyper-methylated and downregulated in the absence of TET1. The epithelial-to-mesenchymal transition phenotype of mutant TSCs is accompanied by centrosome duplication and separation defects. Moreover, we identify a role of TET1 in maintaining cyclin B1 stability, thereby acting as facilitator of mitotic cell-cycle progression. As a result, Tet1/2 mutant TSCs are prone to undergo endoreduplicative cell cycles leading to the formation of polyploid trophoblast giant cells. Taken together, our data reveal essential functions of TET proteins in the trophoblast lineage. : TET proteins are well known for their role in pluripotency. Here, Hemberger and colleagues show that TET1 and TET2 are also critical for maintaining the epithelial integrity of trophoblast stem cells. TET1/2 ensure mitotic cell-cycle progression by stabilizing cyclin B1 and by regulating centrosome organization. These insights reveal the importance of TET proteins beyond their role in epigenome remodeling. Keywords: TET proteins, trophoblast stem cells, cell cycle, endoreduplication, self-renewal, mitosis, trophoblast giant cells, differentiation

  20. Prognostic significance of the number of postoperative intraperitoneal chemotherapy cycles for patients with advanced epithelial ovarian cancer.

    Science.gov (United States)

    Suidan, Rudy S; Zhou, Qin; Iasonos, Alexia; O'Cearbhaill, Roisin E; Chi, Dennis S; Long Roche, Kara C; Tanner, Edward J; Denesopolis, John; Barakat, Richard R; Zivanovic, Oliver

    2015-05-01

    Phase 3 trials have demonstrated a survival advantage for patients with optimally debulked epithelial ovarian cancer who received intravenous (IV) and intraperitoneal (IP) chemotherapy compared with IV therapy alone. This was despite a significant proportion of patients in the IV/IP arms not completing all 6 planned cycles. Our objective was to evaluate the prognostic significance of the number of IV/IP cycles administered. Data were analyzed for all patients with stage III to IV epithelial ovarian cancer who underwent optimal primary cytoreduction followed by 1 or more cycles of IV/IP chemotherapy from January 2005 to July 2011 at our institution. A landmark analysis was performed to associate progression-free survival (PFS) and overall survival (OS) with the number of IV/IP cycles given. We identified 201 patients; 26 (13%) received 1 to 2 cycles of IV/IP chemotherapy, 41 (20%) received 3 to 4 cycles, and 134 (67%) received 5 to 6 cycles. The 5-year PFS for patients who received 1 to 2, 3 to 4, and 5 to 6 cycles was 18%, 29%, and 17%, respectively. The 5-year OS for patients who received 1 to 2, 3 to 4, and 5 to 6 cycles was 44%, 54%, and 57%, respectively. There was no significant difference in PFS (P = 0.31) or OS (P = 0.14) between the 3 groups. The most common reason for discontinuing IV/IP therapy was treatment-related toxicity (77%). Postoperative complications were the most common reason for not initiating IV/IP therapy (42%) in patients who subsequently transitioned to it. We did not detect a significant survival difference between patients who received 1 to 2, 3 to 4, or 5 to 6 IV/IP chemotherapy cycles. Women may still derive a survival benefit if they receive fewer than 6 IV/IP cycles.

  1. Cell Cycle Deregulation in Ewing's Sarcoma Pathogenesis

    Science.gov (United States)

    Kowalewski, Ashley A.; Randall, R. Lor; Lessnick, Stephen L.

    2011-01-01

    Ewing's sarcoma is a highly aggressive pediatric tumor of bone that usually contains the characteristic chromosomal translocation t(11;22)(q24;q12). This translocation encodes the oncogenic fusion protein EWS/FLI, which acts as an aberrant transcription factor to deregulate target genes necessary for oncogenesis. One key feature of oncogenic transformation is dysregulation of cell cycle control. It is therefore likely that EWS/FLI and other cooperating mutations in Ewing's sarcoma modulate the cell cycle to facilitate tumorigenesis. This paper will summarize current published data associated with deregulation of the cell cycle in Ewing's sarcoma and highlight important questions that remain to be answered. PMID:21052502

  2. Cell Cycle Deregulation in Ewing's Sarcoma Pathogenesis

    Directory of Open Access Journals (Sweden)

    Ashley A. Kowalewski

    2011-01-01

    Full Text Available Ewing's sarcoma is a highly aggressive pediatric tumor of bone that usually contains the characteristic chromosomal translocation t(11;22(q24;q12. This translocation encodes the oncogenic fusion protein EWS/FLI, which acts as an aberrant transcription factor to deregulate target genes necessary for oncogenesis. One key feature of oncogenic transformation is dysregulation of cell cycle control. It is therefore likely that EWS/FLI and other cooperating mutations in Ewing's sarcoma modulate the cell cycle to facilitate tumorigenesis. This paper will summarize current published data associated with deregulation of the cell cycle in Ewing's sarcoma and highlight important questions that remain to be answered.

  3. Nanoceria have no genotoxic effect on human lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pierscionek, Barbara K; Yasseen, Akeel A [School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA (United Kingdom); Li, Yuebin; Schachar, Ronald A; Chen, Wei [Department of Physics, University of Texas at Arlington, Arlington, TX 76019 (United States); Colhoun, Liza M, E-mail: b.pierscionek@ulster.ac.uk, E-mail: weichen@uta.edu [Centre for Vision and Vascular Sciences, School of Medicine, Dentistry and Biomedical Sciences, Queen' s University Belfast, Grosvenor Road, Belfast, BT12 6BA (United Kingdom)

    2010-01-22

    There are no treatments for reversing or halting cataract, a disease of the structural proteins in the eye lens, that has associations with other age-related degenerative conditions such as Alzheimer's disease. The incidence of cataract and associated conditions is increasing as the average age of the population rises. Protein folding diseases are difficult to assess in vivo as proteins and their age-related changes are assessed after extraction. Nanotechnology can be used to investigate protein changes in the intact lens as well as for a potential means of drug delivery. Nanoparticles, such as cerium oxide (CeO{sub 2}) which have antioxidant properties, may even be used as a means of treating cataract directly. Prior to use in treatments, nanoparticle genotoxicity must be tested to assess the extent of any DNA or chromosomal damage. Sister chromatid exchanges were measured and DNA damage investigated using the alkaline COMET assay on cultured human lens epithelial cells, exposed to 5 and 10 {mu}g ml{sup -1} of CeO{sub 2} nanoparticles (nanoceria). Nanoceria at these dosages did not cause any DNA damage or significant increases in the number of sister chromatid exchanges. The absence of genotoxic effects on lens cells suggests that nanoceria, in the doses and exposures tested in this study, are not deleterious to the eye lens and have the potential for use in studying structural alterations, in developing non-surgical cataract treatments and in investigating other protein folding diseases.

  4. Graphene-induced apoptosis in lung epithelial cells through EGFR

    Science.gov (United States)

    Tsai, Shih-Ming; Bangalore, Preeti; Chen, Eric Y.; Lu, David; Chiu, Meng-Hsuen; Suh, Andrew; Gehring, Matthew; Cangco, John P.; Garcia, Santiago G.; Chin, Wei-Chun

    2017-07-01

    Expanding interest in nanotechnology applied to electronic and biomedical fields has led to fast-growing development of various nanomaterials. Graphene is a single-atom thick, two-dimensional sheet of hexagonally arranged carbon atoms with unique physical and chemical properties. Recently, graphene has been used in many studies on electronics, photonics, composite materials, energy generation and storage, sensors, and biomedicine. However, the current health risk assessment for graphene has been relatively limited and inconclusive. This study evaluated the toxicity effects of graphene on the airway epithelial cell line BEAS-2B, which represents the first barrier of the human body to interact with airborne graphene particles. Our result showed that graphene can induce the cellular Ca2+ by phospholipase C (PLC) associated pathway by activating epidermal growth factor receptor (EGFR). Subsequently, inositol 1,4,5-triphosphate (IP3) receptors activate the release of Ca2+ from the endoplasmic reticulum (ER) Ca2+ stores. Those Ca2+ signals further trigger the calcium-regulated apoptosis in the cell. Furthermore, the stimulation can cause EGFR upregulation, which have been demonstrated to associate with diseases such as lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular diseases. This study highlights the additional health risk considering that it can function as a contributing factor for other respiratory diseases.

  5. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Xianglu Li

    2009-01-01

    Full Text Available HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

  6. Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

    International Nuclear Information System (INIS)

    Rodríguez-Rigueiro, Teresa; Valladares-Ayerbes, Manuel; Haz-Conde, Mar; Aparicio, Luis A; Figueroa, Angélica

    2011-01-01

    The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells. Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed. Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The

  7. Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia.

    Directory of Open Access Journals (Sweden)

    Simon A W G Dello

    Full Text Available OBJECTIVES: The intermittent Pringle maneuver (IPM is frequently applied to minimize blood loss during liver transection. Clamping the hepatoduodenal ligament blocks the hepatic inflow, which leads to a non circulating (hepatosplanchnic outflow. Also, IPM blocks the mesenteric venous drainage (as well as the splenic drainage with raising pressure in the microvascular network of the intestinal structures. It is unknown whether the IPM is harmful to the gut. The aim was to investigate intestinal epithelial cell damage reflected by circulating intestinal fatty acid binding protein levels (I-FABP in patients undergoing liver resection with IPM. METHODS: Patients who underwent liver surgery received total IPM (total-IPM or selective IPM (sel-IPM. A selective IPM was performed by selectively clamping the right portal pedicle. Patients without IPM served as controls (no-IPM. Arterial blood samples were taken immediately after incision, ischemia and reperfusion of the liver, transection, 8 hours after start of surgery and on the first post-operative day. RESULTS: 24 patients (13 males were included. 7 patients received cycles of 15 minutes and 5 patients received cycles of 30 minutes of hepatic inflow occlusion. 6 patients received cycles of 15 minutes selective hepatic occlusion and 6 patients underwent surgery without inflow occlusion. Application of total-IPM resulted in a significant increase in I-FABP 8 hours after start of surgery compared to baseline (p<0.005. In the no-IPM group and sel-IPM group no significant increase in I-FABP at any time point compared to baseline was observed. CONCLUSION: Total-IPM in patients undergoing liver resection is associated with a substantial increase in arterial I-FABP, pointing to intestinal epithelial injury during liver surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01099475.

  8. Regulation of the cell cycle by irradiation

    International Nuclear Information System (INIS)

    Akashi, Makoto

    1995-01-01

    The molecular mechanism of cell proliferation is extremely complex; deregulation results in neoplastic transformation. In eukaryotes, proliferation of cells is finely regulated through the cell cycle. Studies have shown that the cell cycle is regulated by s series of enzymes known as cyclin-dependent kinases (CDKs). The activities of CDKs are controlled by their association with regulatory subunits, cyclins; the expression of cyclins and the activation of the different cyclin-CDK complexes are required for the cell to cycle. Thus, the cell cycle is regulated by activating and inhibiting phosphorylation of the CDK subunits and this program has internal check points at different stages of the cell cycle. When cells are exposed to external insults such as DNA damaging agents, negative regulation of the cell cycle occurs; arrest in either G1 or G2 stage is induced to prevent the cells from prematurely entering into the next stage before DNA is repaired. Recently, a potent inhibitor of CDKs, which inhibits the phosphorylation of retinoblastoma susceptibility (Rb) gene product by cyclin A-CDK2, cyclin E-CDK2, cyclin D1-CDK4, and cyclin D2-CDK4 complexes has been identified. This protein named WAF1, Sdi1, Cip1, or p21 (a protein of Mr 21,000) contains a p53-binding site in its promoter and studies have reported that the expression of WAF1 was directly regulated by p53; cells with loss of p53 activity due to mutational alteration were unable to induce WAF1. This chapter will be focused on the mechanisms of the cell cycle including inhibitors of CDKs, and the induction of WAF1 by irradiation through a pathway independent of p53 will be also described. (author)

  9. Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

    2016-03-10

    Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

  10. Human amniotic epithelial cells combined with silk fibroin scaffold in the repair of spinal cord injury

    Directory of Open Access Journals (Sweden)

    Ting-gang Wang

    2016-01-01

    Full Text Available Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithelial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the transplant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial cells combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.

  11. A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

    Science.gov (United States)

    Macheiner, Tanja; Kuess, Anna; Dye, Julian; Saxena, Amulya K

    2014-01-01

    The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

  12. Cell Extrusion: A Stress-Responsive Force for Good or Evil in Epithelial Homeostasis.

    Science.gov (United States)

    Ohsawa, Shizue; Vaughen, John; Igaki, Tatsushi

    2018-02-05

    Epithelial tissues robustly respond to internal and external stressors via dynamic cellular rearrangements. Cell extrusion acts as a key regulator of epithelial homeostasis by removing apoptotic cells, orchestrating morphogenesis, and mediating competitive cellular battles during tumorigenesis. Here, we delineate the diverse functions of cell extrusion during development and disease. We emphasize the expanding role for apoptotic cell extrusion in exerting morphogenetic forces, as well as the strong intersection of cell extrusion with cell competition, a homeostatic mechanism that eliminates aberrant or unfit cells. While cell competition and extrusion can exert potent, tumor-suppressive effects, dysregulation of either critical homeostatic program can fuel cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Tungsten-induced carcinogenesis in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Laulicht, Freda; Brocato, Jason; Cartularo, Laura; Vaughan, Joshua; Wu, Feng; Kluz, Thomas; Sun, Hong [Department of Environmental Medicine, New York University Langone Medical Center, Tuxedo, NY 10987 (United States); Oksuz, Betul Akgol [Genome Technology Center, New York University Langone Medical Center, New York, NY 10016 (United States); Shen, Steven [Center for Health Informatics and Bioinformatics, New York University Langone Medical Center, New York, NY 10016 (United States); Peana, Massimiliano; Medici, Serenella; Zoroddu, Maria Antonietta [Department of Chemistry and Pharmacy, University of Sassari, Sassari (Italy); Costa, Max, E-mail: Max.Costa@nyumc.org [Department of Environmental Medicine, New York University Langone Medical Center, Tuxedo, NY 10987 (United States)

    2015-10-01

    Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten's ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans. - Highlights: • Tungsten (W) induces cell transformation and increases migration in vitro. • W increases xenograft growth in nude mice. • W altered the expression of cancer-related genes such as those involved in leukemia. • Some of the dysregulated leukemia genes include, CD74, CTGF, MST4, and HOXB5. • For the first time, data is presented that demonstrates tungsten's carcinogenic potential.

  14. Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture

    International Nuclear Information System (INIS)

    Sadlonova, Andrea; Novak, Zdenek; Johnson, Martin R; Bowe, Damon B; Gault, Sandra R; Page, Grier P; Thottassery, Jaideep V; Welch, Danny R; Frost, Andra R

    2005-01-01

    Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF

  15. Alveolar epithelial type II cells induce T cell tolerance to specific antigen

    DEFF Research Database (Denmark)

    Lo, Bernice; Hansen, Søren; Evans, Kathy

    2008-01-01

    The lungs face the immunologic challenge of rapidly eliminating inhaled pathogens while maintaining tolerance to innocuous Ags. A break in this immune homeostasis may result in pulmonary inflammatory diseases, such as allergies or asthma. The observation that alveolar epithelial type II cells (Type...... II) constitutively express the class II MHC led us to hypothesize that Type II cells play a role in the adaptive immune response. Because Type II cells do not express detectable levels of the costimulatory molecules CD80 and CD86, we propose that Type II cells suppress activation of naive T cells...

  16. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    OpenAIRE

    Liu, Z.; Zhang, P.; Zhou, Y.; Qin, H.; Shen, T.

    2010-01-01

    Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithel...

  17. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    Science.gov (United States)

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  18. Disruption of contact inhibition in rat liver epithelial cells by various types of AhR ligands

    Energy Technology Data Exchange (ETDEWEB)

    Vondracek, J.; Chramostova, K.; Kozubik, A. [Institute of Biophysics, Brno (Czech Republic); Krcmar, P.; Machala, M. [Veterinary Research Institute, Brno (Czech Republic)

    2004-09-15

    The maintenance of a balance between cell gain and cell loss is essential for proper liver function. The exact role of aryl hydrocarbon receptor (AhR) in regulating cell proliferation and apoptosis of liver cells remains unclear, since ligand-dependent activation of AhR has been shown to induce cell cycle arrest, proliferation, differentiation or apoptosis, depending on the cellular model used. AhR can directly interact with retinoblastoma protein in hepatic cells, forming protein complexes that can efficiently block cell cycle progression by inducing G1 arrest, or to induce the expression of inhibitors of cyclin-dependent kinases, such as p271. On the other hand, it has been suggested that AhR could play a stimulatory role in cell proliferation, either directly or by mediating a release from contact inhibition. It is now generally accepted that progenitor cells exist in the liver, are activated in various liver diseases and can form a potential target cell population for both tumor initiating and tumor promoting chemicals4. 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) has been found to release rat liver epithelial cells from contact inhibition by upregulating cyclin A expression and cyclin A/cdk2 activity. Our previous studies have shown that a number of AhR ligands5,6 can stimulate proliferation of confluent of rat liver epithelial ''stem-like'' WB-F344 cells. Such mechanism could play a role in liver tumor promotion. In the present study, we used flavonoid compounds that have been reported to act either as pure agonists, such as beta-naphthoflavone (BNF), or as partial/complete antagonists of AhR - alpha-naphthoflavone (ANF) and 3'-methoxy-4'-nitroflavone (3'M4'NF), in order to investigate effects of AhR agonists/antagonists on confluent rat liver epithelial cells. The present study aimed to investigate the effects of model flavonoids on the release of rat liver epithelial cells from contact inhibition, and on inducibility of

  19. Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

    Science.gov (United States)

    Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

    2014-12-01

    Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

  20. Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

    Science.gov (United States)

    O'Hara, Bethany A; Gee, Gretchen V; Atwood, Walter J; Haley, Sheila A

    2018-04-15

    JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML. IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood

  1. In vivo imaging of the retinal pigment epithelial cells

    Science.gov (United States)

    Morgan, Jessica Ijams Wolfing

    The retinal pigment epithelial (RPE) cells form an important layer of the retina because they are responsible for providing metabolic support to the photoreceptors. Techniques to image the RPE layer include autofluorescence imaging with a scanning laser ophthalmoscope (SLO). However, previous studies were unable to resolve single RPE cells in vivo. This thesis describes the technique of combining autofluorescence, SLO, adaptive optics (AO), and dual-wavelength simultaneous imaging and registration to visualize the individual cells in the RPE mosaic in human and primate retina for the first time in vivo. After imaging the RPE mosaic non-invasively, the cell layer's structure and regularity were characterized using quantitative metrics of cell density, spacing, and nearest neighbor distances. The RPE mosaic was compared to the cone mosaic, and RPE imaging methods were confirmed using histology. The ability to image the RPE mosaic led to the discovery of a novel retinal change following light exposure; 568 nm exposures caused an immediate reduction in autofluorescence followed by either full recovery or permanent damage in the RPE layer. A safety study was conducted to determine the range of exposure irradiances that caused permanent damage or transient autofluorescence reductions. Additionally, the threshold exposure causing autofluorescence reduction was determined and reciprocity of radiant exposure was confirmed. Light exposures delivered by the AOSLO were not significantly different than those delivered by a uniform source. As all exposures tested were near or below the permissible light levels of safety standards, this thesis provides evidence that the current light safety standards need to be revised. Finally, with the retinal damage and autofluorescence reduction thresholds identified, the methods of RPE imaging were modified to allow successful imaging of the individual cells in the RPE mosaic while still ensuring retinal safety. This thesis has provided a

  2. Nanoparticle-mediated delivery of the antimicrobial peptide plectasin against Staphylococcus aureus in infected epithelial cells

    DEFF Research Database (Denmark)

    Water, Jorrit Jeroen; Smart, Simon; Franzyk, Henrik

    2015-01-01

    intracellularly in Calu-3 epithelial cells and in THP-1 cells, whereas A549 cells did not show significant uptake of nanoparticles. Overall, encapsulation of plectasin into PLGA-based nanoparticles appears to be a viable strategy to improve the efficacy of plectasin against infections in epithelial tissues....... epithelial cells might thus be a promising approach to combat such infections. In this work, plectasin, which is a cationic AMP of the defensin class, was encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanoparticles using the double emulsion solvent evaporation method. The nanoparticles displayed...... high plectasin encapsulation efficiency (71-90%) and mediated release of the peptide over 24h. The antimicrobial efficacy of the peptide-loaded nanoparticles was investigated using bronchiolar epithelial Calu-3 cell monolayers infected with S. aureus. The plectasin-loaded nanoparticles displayed...

  3. γδ T cells in homeostasis and host defence of epithelial barrier tissues.

    Science.gov (United States)

    Nielsen, Morten M; Witherden, Deborah A; Havran, Wendy L

    2017-12-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body - namely, the epidermis and the intestine - and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

  4. Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

    International Nuclear Information System (INIS)

    Kikuta, Kazuhiro; Masamune, Atsushi; Watanabe, Takashi; Ariga, Hiroyuki; Itoh, Hiromichi; Hamada, Shin; Satoh, Kennichi; Egawa, Shinichi; Unno, Michiaki; Shimosegawa, Tooru

    2010-01-01

    Research highlights: → Recent studies have shown that pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. → Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and scattered, fibroblast-like appearance. → PSCs decreased the expression of epithelial markers but increased that of mesenchymal markers, along with increased migration. → This study suggests epithelial-mesenchymal transition as a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti

  5. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Feng; Jordan, Ashley; Kluz, Thomas [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States); Shen, Steven [Center for Health Informatics and Bioinformatics, New York University Langone Medical Center, New York, NY 10016 (United States); Sun, Hong; Cartularo, Laura A. [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States); Costa, Max, E-mail: Max.Costa@nyumc.org [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States)

    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. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.

  6. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A.; Costa, Max

    2016-01-01

    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. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.

  7. Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

    Science.gov (United States)

    Sasaki, Ayana; Nagatake, Takahiro; Egami, Riku; Gu, Guoqiang; Takigawa, Ichigaku; Ikeda, Wataru; Nakatani, Tomoya; Kunisawa, Jun; Fujita, Yasuyuki

    2018-04-24

    Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector

    Directory of Open Access Journals (Sweden)

    Lauro Augusto de Oliveira

    2010-10-01

    Full Text Available PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS. We evaluated the transduction efficiency (TE over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells. RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1% and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

  9. Preparing nuclei from cells in monolayer cultures suitable for counting and for following synchronized cells through the cell cycle.

    Science.gov (United States)

    Butler, W B

    1984-08-15

    A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.

  10. Interaction of chitin/chitosan with salivary and other epithelial cells-An overview.

    Science.gov (United States)

    Patil, Sharvari Vijaykumar; Nanduri, Lalitha S Y

    2017-11-01

    Chitin and its deacetylated form, chitosan, have been widely used for tissue engineering of both epithelial and mesenchymal tissues. Epithelial cells characterised by their sheet-like tight cellular arrangement and polarised nature, constitute a major component in various organs and play a variety of roles including protection, secretion and maintenance of tissue homeostasis. Regeneration of damaged epithelial tissues has been studied using biomaterials such as chitin, chitosan, hyaluronan, gelatin and alginate. Chitin and chitosan are known to promote proliferation of various embryonic and adult epithelial cells. However it is not clearly understood how this activity is achieved or what are the mechanisms involved in the chitin/chitosan driven proliferation of epithelial cells. Mechanistic understanding of influence of chitin/chitosan on epithelial cells will guide us to develop more targeted regenerative scaffold/hydrogel systems. Therefore, current review attempts to elicit a mechanistic insight into how chitin and chitosan interact with salivary, mammary, skin, nasal, lung, intestinal and bladder epithelial cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Cell cycle arrest induced by radiation

    International Nuclear Information System (INIS)

    Okaichi, Yasuo; Matsumoto, Hideki; Ohnishi, Takeo

    1994-01-01

    It is known that various chemical reactions, such as cell cycle arrest, DNA repair and cell killing, can occur within the cells when exposed to ionizing radiation and ultraviolet radiation. Thus protein dynamics involved in such chemical reactions has received considerable attention. In this article, cell cycle regulation is first discussed in terms of the G2/M-phase and the G1/S-phase. Then, radiation-induced cell cycle arrest is reviewed. Cell cycle regulation mechanism involved in the G2 arrest, which is well known to occur when exposed to radiation, has recently been investigated using yeasts. In addition, recent study has yielded a noticeable finding that the G1 arrest can occur with intracellular accumulation of p53 product following ionization radiation. p53 is also shown to play an extremely important role in both DNA repair and cell killing due to DNA damage. Studies on the role of genes in protein groups induced by radiation will hold promise for the elucidation of cell cycle mechanism. (N.K.) 57 refs

  12. 2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells.

    OpenAIRE

    Andreassen, P R; Margolis, R L

    1992-01-01

    BHK cells blocked at any of several points in the cell cycle override their drug-induced arrest and proceed in the cycle when exposed concurrently to the protein kinase inhibitor 2-aminopurine (2-AP). For cells arrested at various points in interphase, 2-AP-induced cell cycle progression is made evident by arrival of the drug-treated cell population in mitosis. Cells that have escaped from mimosine G1 arrest, from hydroxyurea or aphidicolin S-phase arrest, or from VM-26-induced G2 arrest subs...

  13. Subtotal Ablation of Parietal Epithelial Cells Induces Crescent Formation

    Science.gov (United States)

    Sicking, Eva-Maria; Fuss, Astrid; Uhlig, Sandra; Jirak, Peggy; Dijkman, Henry; Wetzels, Jack; Engel, Daniel R.; Urzynicok, Torsten; Heidenreich, Stefan; Kriz, Wilhelm; Kurts, Christian; Ostendorf, Tammo; Floege, Jürgen; Smeets, Bart

    2012-01-01

    Parietal epithelial cells (PECs) of the renal glomerulus contribute to the formation of both cellular crescents in rapidly progressive GN and sclerotic lesions in FSGS. Subtotal transgenic ablation of podocytes induces FSGS but the effect of specific ablation of PECs is unknown. Here, we established an inducible transgenic mouse to allow subtotal ablation of PECs. Proteinuria developed during doxycycline-induced cellular ablation but fully reversed 26 days after termination of doxycycline administration. The ablation of PECs was focal, with only 30% of glomeruli exhibiting histologic changes; however, the number of PECs was reduced up to 90% within affected glomeruli. Ultrastructural analysis revealed disruption of PEC plasma membranes with cytoplasm shedding into Bowman’s space. Podocytes showed focal foot process effacement, which was the most likely cause for transient proteinuria. After >9 days of cellular ablation, the remaining PECs formed cellular extensions to cover the denuded Bowman’s capsule and expressed the activation marker CD44 de novo. The induced proliferation of PECs persisted throughout the observation period, resulting in the formation of typical cellular crescents with periglomerular infiltrate, albeit without accompanying proteinuria. In summary, subtotal ablation of PECs leads the remaining PECs to react with cellular activation and proliferation, which ultimately forms cellular crescents. PMID:22282596

  14. Expression of human telomerase reverse transcriptase protein in oral epithelial dysplasia and oral squamous cell carcinoma: An immunohistochemical study

    Science.gov (United States)

    Raghunandan, Bangalore Nagarajachar; Sanjai, Karpagaselvi; Kumaraswamy, Jayalakshmi; Papaiah, Lokesh; Pandey, Bhavna; Jyothi, Bellur MadhavaRao

    2016-01-01

    Background: Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA sequences and almost universally provides the molecular basis for unlimited proliferative potential. The telomeres become shorter with each cycle of replication and reach a critical limit; most cells die or enter stage of replicative senescence. Telomere length maintenance by telomerase is required for all the cells that exhibit limitless replicative potential. It has been postulated that reactivation of telomerase expression is necessary for the continuous proliferation of neoplastic cells to attain immortality. Use of immunohistochemistry (IHC) is a useful, reliable method of localizing the human telomerase reverse transcriptase (hTERT) protein in tissue sections which permits cellular localization. Although there exists a lot of information on telomerase in oral cancer, little is known about their expression in oral epithelial dysplasia and their progression to oral squamous cell carcinoma (OSCC) compared to normal oral mucosa. This study addresses this lacuna. Aims: To compare the expression of hTERT protein in oral epithelial dysplasia and OSCC with normal oral mucosa by Immunohistochemical method. Subjects and Methods: In this preliminary study, IHC was used to detect the expression of hTERT protein in OSCC (n = 20), oral epithelial dysplasia (n = 21) and normal oral mucosa (n = 10). The tissue localization of immunostain, cellular localization of immunostain, nature of stain, intensity of stain, percentage of cells stained with hTERT protein were studied. A total number of 100 cells were counted in each slide. Statistical Analysis: All the data were analyzed using SPSS software version 16.0. The tissue localization, cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity was compared across the groups using Pearson's Chi-square test. The mean percentage of cells stained for oral epithelial dysplasia, OSCC and normal oral mucosa were

  15. Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells

    OpenAIRE

    Hombauer, H; Minguell, J J

    2000-01-01

    This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high...

  16. Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells

    International Nuclear Information System (INIS)

    Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

    2010-01-01

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ∼4-fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

  17. TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs

    Directory of Open Access Journals (Sweden)

    Shingo Suzuki

    2016-01-01

    Full Text Available Cystic fibrosis (CF is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ≃100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

  18. Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis.

    Science.gov (United States)

    Mulder, Daniel J; Pooni, Aman; Mak, Nanette; Hurlbut, David J; Basta, Sameh; Justinich, Christopher J

    2011-02-01

    Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. Endothelial induced EMT in breast epithelial cells with stem cell properties

    DEFF Research Database (Denmark)

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla

    2011-01-01

    endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression...... of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D......492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close...

  20. Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Young-Chae Kim

    Full Text Available The androgen receptor (AR mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT. However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR, and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN1A overexpression induced cell cycle arrest. We therefore propose that AR-mediated growth suppression of HPr-1AR involves cyclin D1 mRNA decay, transcriptional repression of cyclin D2 and CDK4/6, and transcriptional activation of CDKN1A, which serve to decrease CDK4/6 activity. AR-mediated inhibition of PC3-Lenti-AR proliferation occurs through a similar mechanism, albeit without down-regulation of cyclin D. Our findings provide insight into AR-mediated regulation of prostate epithelial cell proliferation.

  1. Indian hedgehog regulates intestinal stem cell fate through epithelial-mesenchymal interactions during development

    NARCIS (Netherlands)

    Kosinski, C.; Stange, D.E.; Xu, C.; Chan, A.S.; Ho, C.; Yuen, S.T.; Mifflin, R.C.; Powell, D.W.; Clevers, H.; Leung, S.Y.; Chen, X.N.

    2010-01-01

    BACKGROUND & AIMS: Intestinal stem cells (ISCs) are regulated by the mesenchymal environment via physical interaction and diffusible factors. We examined the role of Indian hedgehog (Ihh) in mesenchymal organization and the mechanisms by which perturbations in epithelial-mesenchymal interactions

  2. Detonation Nanodiamond Toxicity in Human Airway Epithelial Cells Is Modulated by Air Oxidation

    Science.gov (United States)

    Detonational nanodiamonds (DND), a nanomaterial with an increasing range of industrial and biomedical applications, have previously been shown to induce a pro-inflammatory response in cultured human airway epithelial cells (HAEC). We now show that surface modifications induced by...

  3. Xenobiotic Modulation of Human Mammary Epithelial Cell Gap junctional Intercellular Communication and Growth

    National Research Council Canada - National Science Library

    Ruch, Randall

    1998-01-01

    ...), phthalate esters, and dioxin have been implicated in this increase. Many xenobiotics such as DDT and PCBs have weak estrogenic activity and may enhance breast cancer formation by an estrogenic effect on breast epithelial cell growth...

  4. Xenobiotic Modulation of Human Mammary Epithelial Cell Gap Junctional Intercellular Communication and Growth

    National Research Council Canada - National Science Library

    Ruch, Randall

    1997-01-01

    ...), phthalate esters, and dioxin have been implicated in this increase. Many xenobiotics such as DDT and PCBs have weak estrogenic activity and may enhance breast cancer formation by an estrogenic effect on breast epithelial cell growth...

  5. Epithelial cell proliferative activity of Barrett's esophagus : methodology and correlation with traditional cancer risk markers

    NARCIS (Netherlands)

    Peters, FTM; Ganesh, S; Kuipers, EJ; De Jager-Krikken, A; Karrenbeld, A; Harms, Geert; Sluiter, WJ; Koudstaal, J; Klinkenberg-Knol, EC; Lamers, CBHW; Kleibeuker, JH

    Barrett's esophagus (BE) is a premalignant condition, due to chronic gastroesophageal reflux. Effective antireflux therapy may diminish cancer risk. To evaluate this option an intermediate marker is needed. We developed a methodology for measurement of epithelial cell proliferative activity of

  6. Cytological analysis of the epithelial cells in patients with oral candidiasis.

    Science.gov (United States)

    Loss, Rafael; Sandrin, Rodrigo; França, Beatriz Helena Sottile; de Azevedo-Alanis, Luciana Reis; Grégio, Ana Maria Trindade; Machado, Maria Ângela Naval; de Lima, Antonio Adilson Soares

    2011-07-01

    The aim of this study was to evaluate oral epithelial cells of the oral mucosa infected by Candida albicans using exfoliative cytology. Oral smears were collected from clinically normal-appearing mucosa by liquid-based exfoliative cytology of 60 individuals (30 patients with oral candidiasis and 30 healthy controls matched for age and gender) and analysed for morphologic and cytomorphometric technique. Morphologically, candida-infected epithelial cells exhibited nuclear enlargement, perinuclear rings, discrete orangeophilia, and cytoplasmic vacuoles. The cytomorphometric analysis demonstrated that the cytoplasmic area (CA) of the epithelial cells was diminished in patients undergoing candidiasis as compared to the non-infected controls. In addition, there was an augmentation in nuclear area (NA) and NA/CA area ratio. This study revealed that oral mucosa of patients undergoing candidal infection exhibited significant changes in the size and shape of the oral epithelial cells. © 2010 Blackwell Verlag GmbH.

  7. A hybrid mammalian cell cycle model

    Directory of Open Access Journals (Sweden)

    Vincent Noël

    2013-08-01

    Full Text Available Hybrid modeling provides an effective solution to cope with multiple time scales dynamics in systems biology. Among the applications of this method, one of the most important is the cell cycle regulation. The machinery of the cell cycle, leading to cell division and proliferation, combines slow growth, spatio-temporal re-organisation of the cell, and rapid changes of regulatory proteins concentrations induced by post-translational modifications. The advancement through the cell cycle comprises a well defined sequence of stages, separated by checkpoint transitions. The combination of continuous and discrete changes justifies hybrid modelling approaches to cell cycle dynamics. We present a piecewise-smooth version of a mammalian cell cycle model, obtained by hybridization from a smooth biochemical model. The approximate hybridization scheme, leading to simplified reaction rates and binary event location functions, is based on learning from a training set of trajectories of the smooth model. We discuss several learning strategies for the parameters of the hybrid model.

  8. Quantitative analysis of epithelial cells in urine from men with and without urethritis: implications for studying epithelial: pathogen interactions in vivo

    Directory of Open Access Journals (Sweden)

    Whittington Kate

    2009-07-01

    Full Text Available Abstract Background Epithelial cells in first catch urine (FCU specimens from 87 men with and without urethritis were quantified. Epithelial cells were broadly categorised into transitional and squamous populations using morphological characteristics and immunostaining with anti-pan leukocyte and anti-cytokeratin monoclonal antibodies. Findings The majority (77/87 = 89% of samples contained both transitional (76/87 = 87%; range 1 × 104 – 6 × 105, median 6 × 104 and squamous (57/87 = 66%; range 1 × 104 – 8 × 105, median 2 × 104 epithelial cells. The number of transitional cells correlated with the number of squamous cells (Spearman's rho = 0.697 p Conclusion Further studies are required to explore the complexity of epithelial cell populations in urine. These would provide novel opportunities for studying cellular interactions of C. trachomatis in male urethral infections, about which little is currently known.

  9. Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4.

    Science.gov (United States)

    Todaro, M; Lombardo, Y; Francipane, M G; Alea, M Perez; Cammareri, P; Iovino, F; Di Stefano, A B; Di Bernardo, C; Agrusa, A; Condorelli, G; Walczak, H; Stassi, G

    2008-04-01

    We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.

  10. Quercetogetin protects against cigarette smoke extract-induced apoptosis in epithelial cells by inhibiting mitophagy.

    Science.gov (United States)

    Son, Eun Suk; Kim, Se-Hee; Ryter, Stefan W; Yeo, Eui-Ju; Kyung, Sun Young; Kim, Yu Jin; Jeong, Sung Hwan; Lee, Chang Soo; Park, Jeong-Woong

    2018-04-01

    Recent studies demonstrate that the autophagy-dependent turnover of mitochondria (mitophagy) mediates pulmonary epithelial cell death in response to cigarette smoke extract (CSE) exposure, and contributes to emphysema development in vivo during chronic cigarette smoke (CS)-exposure, although the underlying mechanisms remain unclear. Here, we investigated the role of mitophagy in regulating apoptosis in CSE-exposed human lung bronchial epithelial cells. Furthermore, we investigated the potential of the polymethoxylated flavone antioxidant quercetogetin (QUE) to inhibit CSE-induced mitophagy-dependent apoptosis. Our results demonstrate that CSE induces mitophagy in epithelial cells via mitochondrial dysfunction, and causes increased expression levels of the mitophagy-regulator protein PTEN-induced putative kinase-1 (PINK1) and the mitochondrial fission protein dynamin-1-like protein (DRP-1). CSE induced epithelial cell death and increased the expression of the apoptosis-related proteins cleaved caspase-3, -8 and -9. Caspase-3 activity was significantly increased in Beas-2B cells exposed to CSE, and decreased by siRNA-dependent knockdown of DRP-1. Treatment of epithelial cells with QUE inhibited CSE-induced mitochondrial dysfunction and mitophagy by inhibiting phospho (p)-DRP-1 and PINK1 expression. QUE suppressed mitophagy-dependent apoptosis by inhibiting the expression of cleaved caspase-3, -8 and -9 and downregulating caspase activity in human bronchial epithelial cells. These findings suggest that QUE may serve as a potential therapeutic in CS-induced pulmonary diseases. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Colin R Lickwar

    2017-08-01

    Full Text Available The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development

  12. Action of cholera toxin in the intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with a large number of high affinity binding sites in the cell membrane. Binding of 125 I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected. The response (elevation of cellular cAMP) is linear with time for 40 to 50 min and causes a six- to eight-fold increase over control levels (10 to 15 picomole cAMP/mg cellular protein) at steady state. cAMP and agents that increase cAMP production inhibit Cl - -independent Na + influx into the isolated enterocytes whereas chlorpromazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na + entry. Correlation between cellular cAMP levels and the magnitude of Na + influx provides evidence for a cAMP-mediated control of intestinal Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretion. The effect of cAMP on Na + but not Cl - influx preparations can be partially explained in terms of a cAMP-regulated Na + /H + neutral exchange system. Data on the coupling relationship between Na + transport and the intra- and extracellular pH in the enterocytes show that an amiloride-sensitive electroneutral Na + /H + exchange process occurs. This coupling between Na + and H + is partially inhibited by CT and dbcAMP, suggesting that the Na + /H + exchange may be a cAMP-regulated process. 31 references, 32 figures, 5 tables

  13. Alterations in Helicobacter pylori triggered by contact with gastric epithelial cells

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Johnson

    2012-02-01

    Full Text Available Helicobacter pylori lives within the mucus layer of the human stomach, in close proximity to gastric epithelial cells. While a great deal is known about the effects of H. pylori on human cells and the specific bacterial products that mediate these effects, relatively little work has been done to investigate alterations in H. pylori that may be triggered by bacterial contact with human cells. In this review, we discuss the spectrum of changes in bacterial physiology and morphology that occur when H. pylori is in contact with gastric epithelial cells. Several studies have reported that cell contact causes alterations in H. pylori gene transcription. In addition, H. pylori contact with gastric epithelial cells promotes the formation of pilus-like structures at the bacteria-host cell interface. The formation of these structures requires multiple genes in the cag pathogenicity island, and these structures are proposed to have an important role in the type IV secretion system-dependent process through which CagA enters host cells. Finally, H. pylori contact with epithelial cells can promote bacterial replication and the formation of microcolonies, phenomena that are facilitated by the acquisition of iron and other nutrients from infected cells. In summary, the gastric epithelial cell surface represents an important niche for H. pylori, and upon entry into this niche, the bacteria alter their behavior in a manner that optimizes bacterial proliferation and persistent colonization of the host.

  14. Active Vertex Model for cell-resolution description of epithelial tissue mechanics.

    Science.gov (United States)

    Barton, Daniel L; Henkes, Silke; Weijer, Cornelis J; Sknepnek, Rastko

    2017-06-01

    We introduce an Active Vertex Model (AVM) for cell-resolution studies of the mechanics of confluent epithelial tissues consisting of tens of thousands of cells, with a level of detail inaccessible to similar methods. The AVM combines the Vertex Model for confluent epithelial tissues with active matter dynamics. This introduces a natural description of the cell motion and accounts for motion patterns observed on multiple scales. Furthermore, cell contacts are generated dynamically from positions of cell centres. This not only enables efficient numerical implementation, but provides a natural description of the T1 transition events responsible for local tissue rearrangements. The AVM also includes cell alignment, cell-specific mechanical properties, cell growth, division and apoptosis. In addition, the AVM introduces a flexible, dynamically changing boundary of the epithelial sheet allowing for studies of phenomena such as the fingering instability or wound healing. We illustrate these capabilities with a number of case studies.

  15. Molecular mechanisms involved in casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Lee, E.Y.H.P.; Lee, W.H.; Parry, G.; Bissell, M.J.

    1985-01-01

    Mouse mammary epithelial cells (MMEC) secrete a group of milk-specific proteins including various caseins and whey proteins. Dissociated mammary epithelial cells maintain expression of most of their differentiated functions only if cells are plated on a suitable substratum. Casein production and section, cell morphology, and production of α-lactalbumin have been used as markers to assess the degree of differentiation of mammary cells in culture. The general consensus is that cells express their differentiated properties at high levels and for longer periods of time on such substrata. In this paper, the authors demonstrate that modulation of the expression of caseins by floating collagen gels is manifested at several regulatory points

  16. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Dhananjay M Nawandar

    2015-10-01

    Full Text Available Epstein-Barr virus (EBV is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL in immunosuppressed patients. However, the cellular mechanism(s that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1 promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

  17. The Long and Complicated Relationship between Epstein-Barr Virus and Epithelial Cells.

    Science.gov (United States)

    Hutt-Fletcher, Lindsey M

    2017-01-01

    The roles of epithelial cells in infection and persistence of the Epstein-Barr virus (EBV) have long been difficult to resolve. However, recent developments have reinforced the conclusion that these cells are a major site of virus replication and raised the possibility that, like papillomaviruses, EBV has evolved to take advantage of epithelial differentiation to ensure survival, persistence, and spread. Copyright © 2016 American Society for Microbiology.

  18. Cdc42 regulates epithelial cell polarity and cytoskeletal function during kidney tubule development

    DEFF Research Database (Denmark)

    Elias, Bertha C; Das, Amrita; Parekh, Diptiben V

    2015-01-01

    The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development and main......The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development...

  19. Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

    Science.gov (United States)

    Jang, Jun-Ho; Chand, Hitendra S; Bruse, Shannon; Doyle-Eisele, Melanie; Royer, Christopher; McDonald, Jacob; Qualls, Clifford; Klingelhutz, Aloysius J; Lin, Yong; Mallampalli, Rama; Tesfaigzi, Yohannes; Nyunoya, Toru

    2017-04-01

    The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

  20. Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

    Science.gov (United States)

    Yamamoto, Naoki; Kato, Yoshinao; Sato, Atsushi; Hiramatsu, Noriko; Yamashita, Hiromi; Ohkuma, Mahito; Miyachi, Ei-Ichi; Horiguchi, Masayuki; Hirano, Koji; Kojima, Hajime

    2016-08-01

    In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

  1. Plasticity between Epithelial and Mesenchymal States Unlinks EMT from Metastasis-Enhancing Stem Cell Capacity

    NARCIS (Netherlands)

    Beerling, Evelyne; Seinstra, Daniëlle; de Wit, Elzo; Kester, Lennart; van der Velden, Daphne; Maynard, Carrie; Schäfer, Ronny; van Diest, Paul; Voest, Emile; van Oudenaarden, Alexander; Vrisekoop, Nienke; van Rheenen, Jacco

    2016-01-01

    Forced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT) has been reported to alter metastasis by changing migration and stem cell capacity of tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells

  2. Cell Cycle Regulation of Stem Cells by MicroRNAs.

    Science.gov (United States)

    Mens, Michelle M J; Ghanbari, Mohsen

    2018-06-01

    MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.

  3. Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.

    Directory of Open Access Journals (Sweden)

    Jeanette Wagener

    Full Text Available C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.

  4. Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

    Science.gov (United States)

    Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

    2018-02-01

    Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

  5. Expression of a fms-related oncogene in carcinogen-induced neoplastic epithelial cells

    International Nuclear Information System (INIS)

    Walker, C.; Nettesheim, P.; Barrett, J.C.; Gilmer, T.M.

    1987-01-01

    Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injected into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat aveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of ≅ 4 kilobases. The authors conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor

  6. Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

    Science.gov (United States)

    Cerruti, Benedetta; Puliafito, Alberto; Shewan, Annette M.; Yu, Wei; Combes, Alexander N.; Little, Melissa H.; Chianale, Federica; Primo, Luca; Serini, Guido; Mostov, Keith E.; Celani, Antonio

    2013-01-01

    The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell–cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell–cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis. PMID:24145168

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

    Directory of Open Access Journals (Sweden)

    Mariam El-Ashmawy

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

  8. Fuel cell hybrid taxi life cycle analysis

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, Patricia, E-mail: patricia.baptista@ist.utl.pt [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Ribau, Joao; Bravo, Joao; Silva, Carla [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Adcock, Paul; Kells, Ashley [Intelligent Energy, Charnwood Building, HolywellPark, Ashby Road, Loughborough, LE11 3GR (United Kingdom)

    2011-09-15

    A small fleet of classic London Taxis (Black cabs) equipped with hydrogen fuel cell power systems is being prepared for demonstration during the 2012 London Olympics. This paper presents a Life Cycle Analysis for these vehicles in terms of energy consumption and CO{sub 2} emissions, focusing on the impacts of alternative vehicle technologies for the Taxi, combining the fuel life cycle (Tank-to-Wheel and Well-to-Tank) and vehicle materials Cradle-to-Grave. An internal combustion engine diesel taxi was used as the reference vehicle for the currently available technology. This is compared to battery and fuel cell vehicle configurations. Accordingly, the following energy pathways are compared: diesel, electricity and hydrogen (derived from natural gas steam reforming). Full Life Cycle Analysis, using the PCO-CENEX drive cycle, (derived from actual London Taxi drive cycles) shows that the fuel cell powered vehicle configurations have lower energy consumption (4.34 MJ/km) and CO{sub 2} emissions (235 g/km) than both the ICE Diesel (9.54 MJ/km and 738 g/km) and the battery electric vehicle (5.81 MJ/km and 269 g/km). - Highlights: > A Life Cycle Analysis of alternative vehicle technologies for the London Taxi was performed. > The hydrogen powered vehicles have the lowest energy consumption and CO{sub 2} emissions results. > A hydrogen powered solution can be a sustainable alternative in a full life cycle framework.

  9. Fuel cell hybrid taxi life cycle analysis

    International Nuclear Information System (INIS)

    Baptista, Patricia; Ribau, Joao; Bravo, Joao; Silva, Carla; Adcock, Paul; Kells, Ashley

    2011-01-01

    A small fleet of classic London Taxis (Black cabs) equipped with hydrogen fuel cell power systems is being prepared for demonstration during the 2012 London Olympics. This paper presents a Life Cycle Analysis for these vehicles in terms of energy consumption and CO 2 emissions, focusing on the impacts of alternative vehicle technologies for the Taxi, combining the fuel life cycle (Tank-to-Wheel and Well-to-Tank) and vehicle materials Cradle-to-Grave. An internal combustion engine diesel taxi was used as the reference vehicle for the currently available technology. This is compared to battery and fuel cell vehicle configurations. Accordingly, the following energy pathways are compared: diesel, electricity and hydrogen (derived from natural gas steam reforming). Full Life Cycle Analysis, using the PCO-CENEX drive cycle, (derived from actual London Taxi drive cycles) shows that the fuel cell powered vehicle configurations have lower energy consumption (4.34 MJ/km) and CO 2 emissions (235 g/km) than both the ICE Diesel (9.54 MJ/km and 738 g/km) and the battery electric vehicle (5.81 MJ/km and 269 g/km). - Highlights: → A Life Cycle Analysis of alternative vehicle technologies for the London Taxi was performed. → The hydrogen powered vehicles have the lowest energy consumption and CO 2 emissions results. → A hydrogen powered solution can be a sustainable alternative in a full life cycle framework.

  10. Interleukin-6 promotes the migration and cellular senescence and inhibits apoptosis of human intrahepatic biliary epithelial cells.

    Science.gov (United States)

    Li, Ran; Dong, Juan; Bu, Xiu-Qin; Huang, Yong; Yang, Jing-Yu; Dong, Xuan; Liu, Jie

    2018-02-01

    Biliary epithelial cells (BEC) are closely related to some immune regulatory bile duct diseases. However, the complexity and polymorphism of the morphology and function of bile duct cells have hindered further investigation. Therefore, the aim of this study is to investigate how interleukin-6 (IL-6) affects the migration, cellular senescence, and apoptosis of human intrahepatic biliary epithelial cells (HIBECs). The HIBECs were stimulated by different concentrations of IL-6 (0, 5, 10, 15, and 20 ng/mL, respectively). Transwell assay was performed in order to measure the migration abilities, positive β-Galactosidase staining for the cellular senescence of HIBECs, MTT assay for changes of proliferation after IL-6 treatment and flow cytometry for cell cycle and apoptosis. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were conducted in order to detect the mRNA and protein expressions of epithelial-mesenchymal transition (EMT) markers in HIBECs. In comparison to the 0 ng/mL group, in the 5, 10, 15, and 20 ng/mL groups, a significant increase in the number of migratory HIBECs, proliferation, along with mRNA and protein expressions of EMT markers was observed. While the mRNA and protein expressions of epithelial markers, the number of β-galactosidase positive staining cells, as well as apoptosis rate of HIBECs dramatic decreased. Further, the aforementioned changes were significantly more evident in the 15 and 20 ng/mL groups in comparison to the 5 and 10 ng/mL groups. IL-6 may stimulate EMT, enhance the migration and proliferation, and inhibit apoptosis of HIBECs, thus delaying cellular senescence. © 2017 Wiley Periodicals, Inc.

  11. A nuclear glutathione cycle within the cell cycle.

    Science.gov (United States)

    Diaz Vivancos, Pedro; Wolff, Tonja; Markovic, Jelena; Pallardó, Federico V; Foyer, Christine H

    2010-10-15

    The complex antioxidant network of plant and animal cells has the thiol tripeptide GSH at its centre to buffer ROS (reactive oxygen species) and facilitate cellular redox signalling which controls growth, development and defence. GSH is found in nearly every compartment of the cell, including the nucleus. Transport between the different intracellular compartments is pivotal to the regulation of cell proliferation. GSH co-localizes with nuclear DNA at the early stages of proliferation in plant and animal cells. Moreover, GSH recruitment and sequestration in the nucleus during the G1- and S-phases of the cell cycle has a profound impact on cellular redox homoeostasis and on gene expression. For example, the abundance of transcripts encoding stress and defence proteins is decreased when GSH is sequestered in the nucleus. The functions of GSHn (nuclear GSH) are considered in the present review in the context of whole-cell redox homoeostasis and signalling, as well as potential mechanisms for GSH transport into the nucleus. We also discuss the possible role of GSHn as a regulator of nuclear proteins such as histones and PARP [poly(ADP-ribose) polymerase] that control genetic and epigenetic events. In this way, a high level of GSH in the nucleus may not only have an immediate effect on gene expression patterns, but also contribute to how cells retain a memory of the cellular redox environment that is transferred through generations.

  12. Circadian Gating of Epithelial-to-Mesenchymal Transition in Breast Cancer Cells Via Melatonin-Regulation of GSK3β

    Science.gov (United States)

    Mao, Lulu; Dauchy, Robert T.; Blask, David E.; Slakey, Lauren M.; Xiang, Shulin; Yuan, Lin; Dauchy, Erin M.; Shan, Bin; Brainard, George C.; Hanifin, John P.; Duplessis, Tamika T.; Hill, Steven M.

    2012-01-01

    Disturbed sleep-wake cycle and circadian rhythmicity are associated with cancer, but the underlying mechanisms are unknown. Employing a tissue-isolated human breast xenograft tumor nude rat model, we observed that glycogen synthase kinase 3β (GSK3β), an enzyme critical in metabolism and cell proliferation/survival, exhibits a circadian rhythm of phosphorylation in human breast tumors. Exposure to light-at-night suppresses the nocturnal pineal melatonin synthesis, disrupting the circadian rhythm of GSK3β phosphorylation. Melatonin activates GSK3β by inhibiting the serine-threonine kinase Akt phosphorylation, inducing β-catenin degradation and inhibiting epithelial-to-mesenchymal transition, a fundamental process underlying cancer metastasis. Thus, chronic circadian disruption by light-at-night via occupational exposure or age-related sleep disturbances may contribute to cancer incidence and the metastatic spread of breast cancer by inhibiting GSK3β activity and driving epithelial-to-mesenchymal transition in breast cancer patients. PMID:23002080

  13. Taurine Protects Lens Epithelial Cells Against Ultraviolet B-Induced Apoptosis.

    Science.gov (United States)

    Dayang, Wu; Dongbo, Pang

    2017-10-01

    The massive uptake of compatible osmolytes is a self-protective response shared by lens exposed to hypertonic stress and ultraviolet stress. This study aimed to investigate the protective effects of taurine against ultraviolet B-induced cytotoxicity in the lens epithelial cells. Real-time PCR was used to measure osmolytes transport. Radioimmunoassay was used to measure osmolytes uptake. Cell counting kit-8 assays were used to measure cellular viability. Flow cytometry analysis was used to measure apoptosis level. Compared with normotonic stress, hypertonic stress-induced osmolytes uptake into the lens epithelial cells such as betaine, myoinositol and taurine. UVB exposure increased osmolytes transporter mRNA expression together with osmolytes uptake. Moreover, taurine suppressed UVB-induced cell apoptosis in the lens epithelial cells significantly. The effect of compatible osmolyte taurine on cell survival rate may play an important role in cell resistance and adaption to UVB exposure.

  14. Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells.

    Science.gov (United States)

    Amin, Shayista; Thywissen, Andreas; Heinekamp, Thorsten; Saluz, Hans Peter; Brakhage, Axel A

    2014-07-01

    Aspergillus fumigatus is the most important air-borne pathogenic fungus of humans. Upon inhalation of conidia, the fungus makes close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of conidia of A. fumigatus in the human host. Therefore, by also using uracil-auxotrophic mutant strains, we were able to investigate the interaction of A549 lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, unlike professional phagocytes, our study showed that the presence of conidial dihydroxynaphthalene (DHN) melanin enhanced the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus were able to survive within epithelial cells. This was due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects were not observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia were able to inhibit the extrinsic apoptotic pathway in A549 lung epithelial cells even for longer periods. The anti-apoptotic effect was not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, acted anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  16. Sensitization to epithelial antigens in chronic mucosal inflammatory disease. Characterization of human intestinal mucosa-derived mononuclear cells reactive with purified epithelial cell-associated components in vitro.

    OpenAIRE

    Roche, J K; Fiocchi, C; Youngman, K

    1985-01-01

    To explore the auto-reactive potential of cells infiltrating the gut mucosa in idiopathic chronic inflammatory bowel disease, intestinal lamina propria mononuclear cells (LPMC) were isolated, characterized morphologically and phenotypically, and evaluated for antigen-specific reactivity. The last was assessed by quantitating LPMC cytotoxic capabilities against purified, aqueous-soluble, organ-specific epithelial cell-associated components (ECAC) characterized previously. Enzyme-isolated infla...

  17. Thrombin induces epithelial-mesenchymal transition and collagen production by retinal pigment epithelial cells via autocrine PDGF-receptor signaling.

    Science.gov (United States)

    Bastiaans, Jeroen; van Meurs, Jan C; van Holten-Neelen, Conny; Nagtzaam, Nicole M A; van Hagen, P Martin; Chambers, Rachel C; Hooijkaas, Herbert; Dik, Willem A

    2013-12-19

    De-differentiation of RPE cells into mesenchymal cells (epithelial-mesenchymal transition; EMT) and associated collagen production contributes to development of proliferative vitreoretinopathy (PVR). In patients with PVR, intraocular coagulation cascade activation occurs and may play an important initiating role. Therefore, we examined the effect of the coagulation proteins factor Xa and thrombin on EMT and collagen production by RPE cells. Retinal pigment epithelial cells were stimulated with factor Xa or thrombin and the effect on zonula occludens (ZO)-1, α-smooth muscle actin (α-SMA), collagen, and platelet-derived growth factor (PDGF)-B were determined by real-time quantitative-polymerase chain reaction (RQ-PCR), immunofluorescence microscopy, and HPLC and ELISA for collagen and PDGF-BB in culture supernatants, respectively. PDGF-receptor activation was determined by phosphorylation analysis and inhibition studies using the PDGF-receptor tyrosine kinase inhibitor AG1296. Thrombin reduced ZO-1 gene expression (P production of α-SMA and collagen increased. In contrast to thrombin, factor Xa hardly stimulated EMT by RPE. Thrombin clearly induced PDGF-BB production and PDGF-Rβ chain phosphorylation in RPE. Moreover, AG1296 significantly blocked the effect of thrombin on EMT and collagen production. Our findings demonstrate that thrombin is a potent inducer of EMT by RPE via autocrine activation of PDGF-receptor signaling. Coagulation cascade-induced EMT of RPE may thus contribute to the formation of fibrotic retinal membranes in PVR and should be considered as treatment target in PVR.

  18. Lowe Syndrome protein OCRL1 supports maturation of polarized epithelial cells.

    Directory of Open Access Journals (Sweden)

    Adam G Grieve

    Full Text Available Mutations in the inositol polyphosphate 5-phosphatase OCRL1 cause Lowe Syndrome, leading to cataracts, mental retardation and renal failure. We noted that cell types affected in Lowe Syndrome are highly polarized, and therefore we studied OCRL1 in epithelial cells as they mature from isolated individual cells into polarized sheets and cysts with extensive communication between neighbouring cells. We show that a proportion of OCRL1 targets intercellular junctions at the early stages of their formation, co-localizing both with adherens junctional components and with tight junctional components. Correlating with this distribution, OCRL1 forms complexes with junctional components α-catenin and zonula occludens (ZO-1/2/3. Depletion of OCRL1 in epithelial cells growing as a sheet inhibits maturation; cells remain flat, fail to polarize apical markers and also show reduced proliferation. The effect on shape is reverted by re-expressed OCRL1 and requires the 5'-phosphatase domain, indicating that down-regulation of 5-phosphorylated inositides is necessary for epithelial development. The effect of OCRL1 in epithelial maturation is seen more strongly in 3-dimensional cultures, where epithelial cells lacking OCRL1 not only fail to form a central lumen, but also do not have the correct intracellular distribution of ZO-1, suggesting that OCRL1 functions early in the maturation of intercellular junctions when cells grow as cysts. A role of OCRL1 in junctions of polarized cells may explain the pattern of organs affected in Lowe Syndrome.

  19. Isolation and characterization of a neoplastic epithelial cell line derived from irradiated human submaxillary gland

    International Nuclear Information System (INIS)

    Shirasuna, Kanemitsu; Sato, Mitsunobu; Yura, Yoshiaki; Yanagawa, Tetuo; Kubo, Kazuko

    1979-01-01

    Submaxillary tissues taken from a patient whose oral base was irradiated for squamous cell carcinoma were cultured in order to isolate transformed epithelial cells in vitro. The cells showed a fine structure similar to an intermediate duct cell. When they were transplanted in nude mice, salivary tumors developed. It is epidemiologically known that irradiation induces salivary tumors. In this study, the risk of inducement was revealed and a salivary epithelial cell line was used as a model for the analysis of salivary tumors. (Ichikawa, K.)

  20. Do lipids shape the eukaryotic cell cycle?

    Science.gov (United States)

    Furse, Samuel; Shearman, Gemma C

    2018-01-01

    Successful passage through the cell cycle presents a number of structural challenges to the cell. Inceptive studies carried out in the last five years have produced clear evidence of modulations in the lipid profile (sometimes referred to as the lipidome) of eukaryotes as a function of the cell cycle. This mounting body of evidence indicates that lipids play key roles in the structural transformations seen across the cycle. The accumulation of this evidence coincides with a revolution in our understanding of how lipid composition regulates a plethora of biological processes ranging from protein activity through to cellular signalling and membrane compartmentalisation. In this review, we discuss evidence from biological, chemical and physical studies of the lipid fraction across the cell cycle that demonstrate that lipids are well-developed cellular components at the heart of the biological machinery responsible for managing progress through the cell cycle. Furthermore, we discuss the mechanisms by which this careful control is exercised. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  1. Luminal epithelial cells within the mammary gland can produce basal cells upon oncogenic stress.

    Science.gov (United States)

    Hein, S M; Haricharan, S; Johnston, A N; Toneff, M J; Reddy, J P; Dong, J; Bu, W; Li, Y

    2016-03-17

    In the normal mammary gland, the basal epithelium is known to be bipotent and can generate either basal or luminal cells, whereas the luminal epithelium has not been demonstrated to contribute to the basal compartment in an intact and normally developed mammary gland. It is not clear whether cellular heterogeneity within a breast tumor results from transformation of bipotent basal cells or from transformation and subsequent basal conversion of the more differentiated luminal cells. Here we used a retroviral vector to express an oncogene specifically in a small number of the mammary luminal epithelial cells and tested their potential to produce basal cells during tumorigenesis. This in-vivo lineage-tracing work demonstrates that luminal cells are capable of producing basal cells on activation of either polyoma middle T antigen or ErbB2 signaling. These findings reveal the plasticity of the luminal compartment during tumorigenesis and provide an explanation for cellular heterogeneity within a cancer.

  2. Relationship of Estimated SHIV Acquisition Time Points During the Menstrual Cycle and Thinning of Vaginal Epithelial Layers in Pigtail Macaques.

    Science.gov (United States)

    Kersh, Ellen N; Ritter, Jana; Butler, Katherine; Ostergaard, Sharon Dietz; Hanson, Debra; Ellis, Shanon; Zaki, Sherif; McNicholl, Janet M

    2015-12-01

    HIV acquisition in the female genital tract remains incompletely understood. Quantitative data on biological HIV risk factors, the influence of reproductive hormones, and infection risk are lacking. We evaluated vaginal epithelial thickness during the menstrual cycle in pigtail macaques (Macaca nemestrina). This model previously revealed increased susceptibility to vaginal infection during and after progesterone-dominated periods in the menstrual cycle. Nucleated and nonnucleated (superficial) epithelial layers were quantitated throughout the menstrual cycle of 16 macaques. We examined the relationship with previously estimated vaginal SHIVSF162P3 acquisition time points in the cycle of 43 different animals repeatedly exposed to low virus doses. In the luteal phase (days 17 to cycle end), the mean vaginal epithelium thinned to 66% of mean follicular thickness (days 1-16; P = 0.007, Mann-Whitney test). Analyzing 4-day segments, the epithelium was thickest on days 9 to 12 and thinned to 31% thereof on days 29 to 32, with reductions of nucleated and nonnucleated layers to 36% and 15% of their previous thickness, respectively. The proportion of animals with estimated SHIV acquisition in each cycle segment correlated with nonnucleated layer thinning (Pearson r = 0.7, P layer thinning (Pearson r = 0.6, P = 0.15). These data provide a detailed picture of dynamic cycle-related changes in the vaginal epithelium of pigtail macaques. Substantial thinning occurred in the superficial, nonnucleated layer, which maintains the vaginal microbiome. The findings support vaginal tissue architecture as susceptibility factor for infection and contribute to our understanding of innate resistance to SHIV infection.

  3. Alveolar type II epithelial cell dysfunction in rat experimental hepatopulmonary syndrome (HPS.

    Directory of Open Access Journals (Sweden)

    Wenli Yang

    Full Text Available The hepatopulmonary syndrome (HPS develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2 play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL. We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064 induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.

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

    International Nuclear Information System (INIS)

    William Petersen, Ole; Lind Nielsen, Helga; Gudjonsson, Thorarinn; Villadsen, René; Rønnov-Jessen, Lone; Bissell, Mina J

    2001-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2001-05-12

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

  6. Yongdamsagan-tang, a traditional herbal formula, inhibits cell growth through the suppression of proliferation and inflammation in benign prostatic hyperplasia epithelial-1 cells.

    Science.gov (United States)

    Park, Eunsook; Lee, Mee-Young; Seo, Chang-Seob; Jeon, Woo-Young; Shin, Hyeun-Kyoo

    2017-09-14

    Benign prostatic hyperplasia (BPH), also called benign enlargement of the prostate, is a progressive disease that is observed in most elderly men. Yongdamsagan-tang, a traditional herbal formula, is used commonly for the treatment of inflammation-related diseases. Although the therapeutic efficacy of Yongdamsagan-tang against BPH in vivo was reported previously, its underlying mechanisms are not clearly understood. In this study, we investigated the effect of Yongdamsagan-tang water extract (YSTE) and its mechanism on the growth of human BPH epithelial BPH-1 cells. YSTE was extracted from 11 herbaceous plants and its chemical composition was analyzed by High-performance liquid chromatography (HPLC). YSTE was treated in the epithelial BPH-1 cell line and then cell lysates or supernant were used to evaluate cell viability, cell cycle, proliferation and cytokine production. HPLC revealed that Baicalin and gentiopicroside were involved as the major compounds of YSTE. YSTE treatment in BPH-1 cells repressed cell viability in a dose-dependent manner. Regarding the inhibitory mechanisms of YSTE on cell growth, YSTE inhibited cell proliferation via a decrease in endogenous cyclin D1 protein levels and arrest at the S phase during cell-cycle progression. Furthermore, YSTE treatment in BPH-1 cells suppressed prostaglandin E 2 production and cyclooxygenase-2 (COX-2) protein levels. The secretion of the proinflammatory cytokines, interleukin-8 and interleukin-6, was also reduced by YSTE treatment. YSTE in BPH-1 cells showed antiproliferative and anti-inflammatory activities via cell-cycle arrest and downregulation of COX-2 expression, respectively. Taken together, the results of the present study will enhance our understanding of the mechanisms underlying the effect of YSTE in BPH. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  7. Moderate plasma activated media suppresses proliferation and migration of MDCK epithelial cells

    International Nuclear Information System (INIS)

    Mohades, Soheila; Laroussi, Mounir; Maruthamuthu, Venkat

    2017-01-01

    Low-temperature plasma has been shown to have diverse biomedical uses, including its applications in cancer and wound healing. One recent approach in treating mammalian cells with plasma is through the use of plasma activated media (PAM), which is produced by exposing cell culture media to plasma. While the adverse effects of PAM treatment on cancerous epithelial cell lines have been recently studied, much less is known about the interaction of PAM with normal epithelial cells. In this paper, non-cancerous canine kidney MDCK (Madin-Darby Canine Kidney) epithelial cells were treated by PAM and time-lapse microscopy was used to directly monitor their proliferation and random migration upon treatment. While longer durations of PAM treatment led to cell death, we found that moderate levels of PAM treatment inhibited proliferation in these epithelial cells. We also found that PAM treatment reduced random cell migration within epithelial islands. Immunofluorescence staining showed that while there were no major changes in the actin/adhesion apparatus, there was a significant change in the nuclear localization of proliferation marker Ki-67, consistent with our time-lapse results. (paper)

  8. Membrane associated ion transport enzymes in normal and transformed fibroblasts and epithelial cells

    International Nuclear Information System (INIS)

    Borek, C.

    1982-01-01

    In an effort to evaluate membrane changes associated with neoplastic transformation of fibroblasts and epithelial cells by radiation and chemicals, alterations in membrane-associated (Na + + K + )-ATPase and 5'-nucleotidase activities were investigated. Cell cultures consisted of normal and radiation transformed hamster embryo fibroblasts (HE) and mouse C3H 10T 1/2 fibroblasts, normal and chemically transformed adult rat liver epithelial cells (ARL), as well as hepatocarcinoma cells induced by the liver transformants. Transformed fibroblasts demonstrated a 1-2 fold increase in (Na + + K + )-ATPase activity over the normal, while the transformed liver epithelial cells and carcinoma cells showed a 60% and 40% decrease in activity compared to the normal values, respectively. The 5'-nucleotidase activity was 2 to 3 times higher in the transformed fibroblasts

  9. Response of cultured normal human mammary epithelial cells to X rays

    International Nuclear Information System (INIS)

    Yang, T.C.; Stampfer, M.R.; Smith, H.S.

    1983-01-01

    The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D 0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique

  10. Inhibiting glycogen synthase kinase-3 and transforming growth factor-β signaling to promote epithelial transition of human adipose mesenchymal stem cells.

    Science.gov (United States)

    Setiawan, Melina; Tan, Xiao-Wei; Goh, Tze-Wei; Hin-Fai Yam, Gary; Mehta, Jodhbir S

    2017-09-02

    This study was aimed to investigate the epithelial differentiation of human adipose-derived mesenchymal stem cells (ADSCs) by inhibiting glycogen synthase kinase-3 (GSK3) and transforming growth factor β (TGFβ) signaling. STEMPRO human ADSCs at passage 2 were treated with CHIR99021 (GSK3 inhibitor), E-616452 (TGFβ1 receptor kinase inhibitor), A-83-01 (TGFβ type 1 receptor inhibitor), valproic acid (histone deacetylase inhibitor), tranylcypromine (monoamine oxidase inhibitor) and all-trans retinoic acid for 72 h. The mesenchymal-epithelial transition was shown by down-regulation of mesenchymal genes (Slug, Zinc Finger E-box Binding Homeobox 1 ZEB1, integrin α5 ITGA5 and vimentin VIM) and up-regulation of epithelial genes (E-cadherin, Epithelial Cell Adhesion Molecule EpCAM, Zonula Occludens-1 ZO-1, occludin, deltaN p63 δNp63, Transcription Factor 4 TCF4 and Twist Family bHLH Transcription Factor TWIST), compared to untreated ADSCs. Cell morphology and stress fiber pattern were examined and the treated cells became less migratory in scratch wound closure assay. The formation of cell junction complexes was observed under transmission electron microscopy. Global gene expression using GeneChip ® Human Genome U133 Array (Affymetrix) showed that the treatment up-regulated 540 genes (containing genes for cell cycle, cytoskeleton reorganization, chemotaxis, epithelium development and regulation of cell migration) and down-regulated 483 genes. Human ADSCs were transited to epithelial lineage by inhibiting GSK3 and TGFβ signaling. It can be an adult stem cell source for epithelial cell-based therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

    Directory of Open Access Journals (Sweden)

    Shigenobu Yonemura

    Full Text Available Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D culture systems rather than in two-dimensional (2-D culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules, EpH4 cells (mouse mammary gland, and R2/7 cells (human colon expressing wild-type α-catenin (R2/7 α-Cate cells. These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

  12. Cell cycle kinetics and radiation therapy

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1975-01-01

    Radiation therapy as currently practiced involves the subtle largely empirical art of balancing the recurrence of cancer due to undertreatment against severe damage to local tissues due to overtreatment. Therapeutic results too often fall short of desired success rates; yet, the therapist is continually tantalized to the likelihood that a slight shift of therapeutic ratio favoring normal tissue over cancer would have a profoundly beneficial effect. The application of cell cycle kinetics to radiation therapy is one hope for improving the therapeutic ratio; but, as I will try to show, kinetic approaches are complex, poorly understood, and presently too elusive to elicit confidence or to be used clinically. Their promise lies in their diversity and in the magnitude of our ignorance about how they work and how they should be used. Potentially useful kinetic approaches to therapy can be grouped into three classes. The first class takes advantage of intracyclic differential sensitivity, an effect involving the metabolism and biology of the cell cycle; its strategies are based on synchronization of cells over intervals of hours to days. The second class involves the distinction between cycling and noncycling cells; its strategies are based on the resistance of noncycling cells to cycle-linked radiation sensitizers and chemotherapeutic agents. The third class uses cell repopulation between fractions; its strategies are based on the relative growth rates of tumor and relevant normal tissue before and after perturbation

  13. Cell Cycle Inhibition To Treat Sleeping Sickness

    Directory of Open Access Journals (Sweden)

    Conrad L. Epting

    2017-09-01

    Full Text Available African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei. During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR, which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo. These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.

  14. Early Alterations in Ovarian Surface Epithelial Cells and Induction of Ovarian Epithelial Tumors Trig