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Sample records for apoptotic epithelial cells

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

    PubMed

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

    2016-04-01

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

  2. SHOTGUN PROTEOMICS: IDENTIFICATION OF UNIQUE PROTEIN PROFILES OF APOPTOTIC BODIES FROM BILIARY EPITHELIAL CELLS

    PubMed Central

    Lleo, Ana; Zhang, Weici; McDonald, W. Hayes; Seeley, Erin H.; Leung, Patrick S.C.; Coppel, Ross L.; Ansari, Aftab A.; Adams, David H.; Afford, Simon; Invernizzi, Pietro; Gershwin, M. Eric

    2014-01-01

    Shotgun proteomics is a powerful analytic method to characterize complex protein mixtures in combination with multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). We have used this platform for proteomic characterization of apoptotic bodies in efforts to define the complex protein mixtures found in primary cultures of human intrahepatic biliary epithelial cells (HiBEC), human renal proximal tubular epithelial cells, human bronchial epithelial cells, isolated intrahepatic biliary epithelial cells from explanted primary biliary cirrhosis (PBC) and control liver, using a total of 24 individual samples. Further, as additional controls and for purposes of comparison, proteomic signatures were also obtained from intact cells and apoptotic bodies. The data obtained from LC-MS/MS, combined with database searches and protein assembly algorithms, allowed us to address significant differences in protein spectral counts and identify unique pathways that may be a component to the induction of the signature inflammatory cytokine response against BECs, including the Notch signaling pathway, IL8, IL6, CXCR2 and integrin signaling. Indeed there are 11 proteins that localize specifically to apoptotic bodies of HiBEC and 8 proteins that were specifically absent in HiBEC apoptotic bodies. In conclusion, proteomic analysis of BECs from PBC liver compared to normal liver are significantly different, suggesting that an immunological attack affects the repertoire of proteins expressed and that such cells should be thought of as living in an environment undergoing continuous selection secondary to an innate and adaptive immune response, reflecting an almost “Darwinian” bias. PMID:24841946

  3. Mononuclear phagocytes rapidly clear apoptotic epithelial cells in the proximal epididymis

    PubMed Central

    Smith, T. B.; Cortez-Retamozo, V.; Grigoryeva, L. S.; Hill, E.; Pittet, M. J.; Da Silva, N.

    2015-01-01

    SUMMARY We have shown previously that a network of mononuclear phagocytes (MPs) expressing macrophage and dendritic cell markers such as CD11c, F4/80 and CX3CR1, lines the base of the epididymal tubule. However, in the initial segment (IS) and only in that particular segment, epididymal MPs establish extremely close interactions with the epithelium by projecting slender dendrites between most epithelial cells. We undertook the present study to determine how epididymal phagocytes respond to the transient wave of apoptosis initiated by unilateral efferent duct ligation (EDL) in the epididymal epithelium. We show profound morphological and phenotypical changes restricted to the MPs populating the proximal epididymis following EDL. Within 48 h, a large subset of IS epithelial cells had entered an apoptotic state, visualized by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and CD11c+ and CX3CR1+ MPs readily engulfed TUNEL-positive cells and other debris. Despite the high levels of apoptosis and the rapid clearance of apoptotic cells occurring after EDL, the epithelium preserved its overall architecture and maintained tight junctions of the blood–epididymis barrier (BEB). The discovery of a functional population of MPs in the epididymal epithelium responsible for maintaining the integrity of the BEB raises further questions regarding the role of these cells in clearing defective epithelial cells in the steady-state epididymis, as well as pathogens and abnormal spermatozoa in the lumen. PMID:25082073

  4. Serotonin activates cell survival and apoptotic death responses in cultured epithelial thyroid cells.

    PubMed

    Cerulo, Giuliana; Tafuri, Simona; De Pasquale, Valeria; Rea, Silviana; Romano, Simona; Costagliola, Anna; Della Morte, Rossella; Avallone, Luigi; Pavone, Luigi Michele

    2014-10-01

    Anatomic and physiological interactions between central serotonergic system and thyroid gland are well established. However, the effects of locally available serotonin on the thyroid functions are poorly known. Here, we first demonstrate the expression of serotonin transporter SERT and 5-HT2A receptor subtype in rat thyroid epithelial cell line FRT both at mRNA and protein levels. In order to investigate the molecular mechanisms of serotonin action, FRT cells were exposed to increasing concentrations of the amine. Low concentrations of serotonin (up to 5 μM) enhanced FRT cell growth, and ERK1/2 and SMAD2/3 phosphorylation. Cell exposure to the selective 5-HT2A receptor agonist DOI recapitulated the effects of 5-HT on ERK1/2 phosphorylation. By contrast, administration of M100907, a specific 5-HT2A receptor inhibitor, prevented 5-HT induced ERK1/2 activation. On the other hand, high doses of serotonin (50 μM up to 1 mM) activated a caspase-3 mediated apoptosis of cells. Overall, our findings demonstrate that low levels of serotonin, interacting with 5-HT2A receptor, are able to activate proliferative signals in the thyroid epithelial cells, while high levels of serotonin cause pro-apoptotic responses, thus suggesting an active role of the amine in the thyroid functions and disorders. PMID:24997405

  5. Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages.

    PubMed

    Hodge, S; Hodge, G; Brozyna, S; Jersmann, H; Holmes, M; Reynolds, P N

    2006-09-01

    Chronic obstructive pulmonary disease (COPD) is associated with increased apoptosis and defective phagocytosis in the airway. As uncleared cells can undergo secondary necrosis and perpetuate inflammation, strategies to improve clearance would have therapeutic significance. There is evidence that the 15-member macrolide antibiotic azithromycin has anti-inflammatory properties. Its effects may be increased in the lung due to its ability to reach high concentrations in alveolar macrophages (AMs). The present study investigated the effects of low-dose (500 ng x mL(-1)) azithromycin on the phagocytosis of apoptotic bronchial epithelial cells and neutrophils by AMs. Flow cytometry was applied to measure phagocytosis and receptors involved in AM recognition of apoptotic cells. Cytokines were investigated using cytometric bead array. Baseline phagocytosis was reduced in COPD subjects compared with controls. Azithromycin significantly improved the phagocytosis of epithelial cells or neutrophils by AMs from COPD subjects by 68 and 38%, respectively, often up to levels comparable with controls. The increase in phagocytosis was partially inhibited by phosphatidylserine, implicating the phosphatidylserine pathway in the pro-phagocytic effects of azithromycin. Azithromycin had no effect on other recognition molecules (granulocyte-macrophage colony-stimulating factor, CD44, CD31, CD36, CD91, alphavbeta3 integrin). At higher doses, azithromycin decreased levels of pro-inflammatory cytokines. Thus, low-dose azithromycin therapy could provide an adjunct therapeutic option in chronic obstructive pulmonary disease. PMID:16737992

  6. Thimerosal induces apoptotic and fibrotic changes to kidney epithelial cells in vitro.

    PubMed

    Carneiro, Maria Fernanda Hornos; Morais, Christudas; Small, David M; Vesey, David A; Barbosa, Fernando; Gobe, Glenda C

    2015-12-01

    Thimerosal is an ethyl mercury-containing compound used mainly in vaccines as a bactericide. Although the kidney is a key target for mercury toxicity, thimerosal nephrotoxicity has not received the same attention as other mercury species. The aim of this study was to determine the potential cytotoxic mechanisms of thimerosal on human kidney cells. Human kidney proximal tubular epithelial (HK2) cells were exposed for 24 h to thimerosal (0-2 µM), and assessed for cell viability, apoptosis, and cell proliferation; expression of proteins Bax, nuclear factor-κB subunits, and transforming growth factor-β1 (TGFβ1); mitochondrial health (JC-1, MitoTracker Red CMXRos); and fibronectin levels (enzyme-linked immunosorbent assay). Thimerosal diminished HK2 cell viability and mitosis, promoted apoptosis, impaired the mitochondrial permeability transition, enhanced Bax and TGFβ1 expression, and augmented fibronectin secretion. This is the first report about kidney cell death and pro-fibrotic mechanisms promoted by thimerosal. Collectively, these in vitro results demonstrate that (1) thimerosal induces kidney epithelial cell apoptosis via upregulating Bax and the mitochondrial apoptotic pathway, and (2) thimerosal is a potential pro-fibrotic agent in human kidney cells. We suggest that new evidence on toxicity as well as continuous surveillance in terms of fibrogenesis is required concerning thimerosal use. PMID:24942245

  7. Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF.

    PubMed

    Yoon, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Park, Young Mi; Kang, Jihee Lee

    2016-01-01

    Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors. PMID:26875548

  8. Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF

    PubMed Central

    Yoon, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Park, Young Mi; Kang, Jihee Lee

    2016-01-01

    Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors. PMID:26875548

  9. EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival

    PubMed Central

    Kang, Dong; Skalsky, Rebecca L.; Cullen, Bryan R.

    2015-01-01

    Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that can give rise to cancers of both B-cell and epithelial cell origin. In EBV-induced cancers of epithelial origin, including nasopharyngeal carcinomas (NPCs) and gastric carcinomas, the latent EBV genome expresses very high levels of a cluster of 22 viral pre-miRNAs, called the miR-BARTs, and these have previously been shown to confer a degree of resistance to pro-apoptotic drugs. Here, we present an analysis of the ability of individual miR-BART pre-miRNAs to confer an anti-apoptotic phenotype and report that five of the 22 miR-BARTs demonstrate this ability. We next used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to globally identify the mRNA targets bound by these miR-BARTs in latently infected epithelial cells. This led to the identification of ten mRNAs encoding pro-apoptotic mRNA targets, all of which could be confirmed as valid targets for the five anti-apoptotic miR-BARTs by indicator assays and by demonstrating that ectopic expression of physiological levels of the relevant miR-BART in the epithelial cell line AGS resulted in a significant repression of the target mRNA as well as the encoded protein product. Using RNA interference, we further demonstrated that knockdown of at least seven of these cellular miR-BART target transcripts phenocopies the anti-apoptotic activity seen upon expression of the relevant EBV miR-BART miRNA. Together, these observations validate previously published reports arguing that the miR-BARTs can exert an anti-apoptotic effect in EBV-infected epithelial cells and provide a mechanistic explanation for this activity. Moreover, these results identify and validate a substantial number of novel mRNA targets for the anti-apoptotic miR-BARTs. PMID:26070070

  10. Proteolytic activation of latent TGF-beta precedes caspase-3 activation and enhances apoptotic death of lung epithelial cells.

    PubMed

    Solovyan, Victor T; Keski-Oja, Jorma

    2006-05-01

    Transforming growth factors beta (TGF-betas) are multifunctional cytokines, which are secreted in latent forms in large latent TGF-beta complexes (LL-TGF-beta) with subsequent deposition to the extracellular matrix (ECM). While a variety of mechanisms capable of activating latent TGF-beta in vitro have been described, the physiological conditions, which promote the activation of TGF-beta in vivo are poorly understood. Mink lung epithelial cells (Mv1Lu) are a widely used model for evaluation of the effects of exogenous TGF-beta both in transcriptional and growth inhibitor assays. We find here that apoptosis of Mv1Lu cells, induced either by staurosporine or serum deprivation, is accompanied by proteolytic processing of LL-TGF-beta and the activation of endogenous TGF-beta. Activation of TGF-beta preceded caspase-3 activation and was almost completely suppressed by the serine protease inhibitor, AEBSF. Both exogenous and endogenously activated TGF-betas were able to enhance the apoptotic response of Mv1Lu cells leading to potentiation of cell death. Potentiation of cell death by activated TGF-beta was associated with downregulation of Akt and p38 MAPK, which were both activated at the initial stages of Mv1Lu apoptosis and were suppressed by exogenous TGF-beta. Pharmacological interruption of either phosphoinositide-3-kinase (PI-3K)/Akt or p38 MAPK signaling by the specific inhibitors mimicked the effect of TGF-beta leading to potentiation of cell death. Current results suggest that proteolytic activation of endogenous TGF-beta is a component of the apoptotic response, capable of modulating the death of Mv1Lu cells by inhibition of both PI-3K/Akt and p38 MAPK-dependent survival pathways. PMID:16447253

  11. Brucella suis vaccine strain S2-infected immortalized caprine endometrial epithelial cell lines induce non-apoptotic ER-stress.

    PubMed

    Wang, Xiangguo; Lin, Pengfei; Yin, Yanlong; Zhou, Jinhua; Lei, Lanjie; Zhou, Xudong; Jin, Yaping; Wang, Aihua

    2015-05-01

    Brucella, which is regarded as an intracellular pathogen responsible for a zoonotic disease called brucellosis, survives and proliferates within several types of phagocytic and non-phagocytic cells. Brucella infects not only their preferred hosts but also other domestic and wild animal species, inducing abortion and infertility. Therefore, the interaction between uterine cells and Brucella is important for understanding the pathogenesis of this disease. In this study, we describe the Brucella suis vaccine strain S2 (B.suis.S2) infection and replication in the immortalized caprine endometrial epithelial cell line hTERT-EECs and the induced cellular and molecular response modulation in vitro. We found that B.suis S2 was able to infect and replicate to high titers and inhibit the proliferation of EECs and induce non-apoptotic pathways, as determined by B.suis.S2 detection using MTT and acridine orange/ethidium bromide (AO/EB) staining and flow cytometry. We explored the evidence of non-apoptotic pathways using real-time quantitative RT-PCR and by western blot analysis. Finally, we discovered the over-expression of GRP78, ATF4, ATF6, PERK, eIF2α, CHOP, and cytochrome c (Cyt-c) but not IRE1, xbp-1, and caspase-3 in B.suis.S2 (HK)-attacked and B.suis.S2-infected cells, suggesting that the molecular mechanism of ER stress sensor activation by B.suis.S2 is basically concomitant with that by B.suis.S2 (HK) and that ER stress, especially the PERK pathway, plays an important role in the process of B.suis.S2 infecting EEC, which may, in part, explain the role of the uterus in the pathogenesis of B.suis.S2. PMID:25633898

  12. Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells

    PubMed Central

    Das, Soumita; Sarkar, Arup; Ryan, Kieran A.; Fox, Sarah; Berger, Alice H.; Juncadella, Ignacio J.; Bimczok, Diane; Smythies, Lesley E.; Harris, Paul R.; Ravichandran, Kodi S.; Crowe, Sheila E.; Smith, Phillip D.; Ernst, Peter B.

    2014-01-01

    After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects.—Das, S., Sarkar, A., Ryan, K. A., Fox, S., Berger, A. H., Juncadella, I. J., Bimczok, D., Smythies, L. E., Harris, P. R., Ravichandran, K. S., Crowe, S. E., Smith, P. D., Ernst, P. B. Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells. PMID:24509909

  13. Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions.

    PubMed

    Soriano-Romaní, Laura; García-Posadas, Laura; López-García, Antonio; Paraoan, Luminita; Diebold, Yolanda

    2015-05-01

    Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-β2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-β2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down

  14. Caffeic acid phenethyl ester activates pro-apoptotic and epithelial-mesenchymal transition-related genes in ovarian cancer cells A2780 and A2780cis.

    PubMed

    Gherman, Claudia; Braicu, Ovidiu Leonard; Zanoaga, Oana; Jurj, Anca; Pileczki, Valentina; Maralani, Mahafarin; Drigla, Flaviu; Braicu, Cornelia; Budisan, Liviuta; Achimas-Cadariu, Patriciu; Berindan-Neagoe, Ioana

    2016-02-01

    Ovarian cancer is a highly aggressive pathology, displaying a poor prognosis and chemoresistance to classical therapy. The present study was conducted to evaluate the effect of caffeic acid phenethyl ester (CAPE) on survival of ovarian cancer cell lines, A2780 (sensitive to cisplatin) and A2780cis (resistant to cisplatin). MTT assay was used to evaluate cell viability, while the apoptotic processes were examined by flow cytometry and qRT-PCR. A reduction of cell proliferation and activation of the apoptosis was observed in both cell lines. qRT-PCR evaluation demonstrated the activation of the pro-apoptotic genes (BAD, CASP8, FAS, FADD, p53) in both cell lines. The limited therapeutic effect in A2780 cells is explained by the activation of epithelial-mesenchymal transition-related genes (ZEB1, ZEB2, or TGFBB1) as displayed by Ingenuity Network analysis. Overall data suggest that CAPE can be used as an alternative in sensitizing cells to chemotherapy. PMID:26838168

  15. Comparative Effect of Recombinant Shiga Toxin in Induction of Pro- and Anti-Apoptotic Markers and Inflammatory Cytokines in Epithelial and Monocytic Cells

    PubMed Central

    Abedi Jafari, Fatemeh; Oloomi, Mana; Bouzari, Saeid

    2016-01-01

    Background: Shiga toxins (Stxs, also referred to as verotoxins) are a family of bacterial protein toxins generated by Stx producing-Escherichia coli (STEC), such as E. coli serotype O157:H7. Objectives: The aim of this study was to investigate the effect of recombinant and native Shiga toxin (Stx) in induction of pro- and anti-apoptosis factors and stimulation of immune response to HeLa and THP-1 cells. Materials and Methods: The HeLa and THP-1 cells were used to study the effect of native and recombinant Shiga toxin. For this purpose, 106 cells were cultured overnight in six-well plates and different concentrations of Stx were added to each well. The cells were then collected after 24 hours of incubation. Total RNA and protein was extracted. Firstly, the total RNA was used in reverse transcription-polymerase chain reaction (RT-PCR) for detection of interleukin (IL)-1α, IL-1β, IL-8, tumor necrosis factor (TNF)-α, B-cell lymphoma (Bcl)-2 and Bcl-xl transcript. Protein expression of pro- and anti-apoptotic factors was also confirmed by western blot analysis. Results: The IL-1α and IL-8 were increased by recombinant and native Stx. Interleukin-1β was detected in THP-1, while TNF-α was detected HeLa cells. Furthermore, Bcl-2 and Bcl-xl expression was observed in HeLa cells. However, expression of Bak was reduced by recombinant Stx and native toxin at the protein level, while Bcl-xl expression was increased. Conclusions: These results suggest that toxins induce inflammatory responses, particularly through expression of chemokine. Recombinant Stx and native toxin induced apoptosis by balancing between different pro- and anti-apoptotic Bcl-2 family-factors in epithelial cells. In this study, for the first time, recombinant and native Stx induction of apoptotic factors and stimulation of immune response to HeLa and THP-1 cells were compared. PMID:27127585

  16. Immunosuppressive effects of apoptotic cells

    NASA Astrophysics Data System (ADS)

    Voll, Reinhard E.; Herrmann, Martin; Roth, Edith A.; Stach, Christian; Kalden, Joachim R.; Girkontaite, Irute

    1997-11-01

    Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

  17. Cytotoxicity of carteolol to human corneal epithelial cells by inducing apoptosis via triggering the Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway.

    PubMed

    Shan, Ming; Fan, Ting-Jun

    2016-09-01

    Carteolol is a frequently used nonselective β-adrenoceptor antagonist for glaucoma and ocular hypertension treatment, and its repeated/prolonged usage might be cytotoxic to the cornea, especially the outmost human corneal epithelium (HCEP). The aim of the present study was to characterize the cytotoxicity of carteolol to HCEP and its underlying cellular and molecular mechanisms using an in vitro model of HCEP cells. After HCEP cells were treated with carteolol at concentrations varying from 2% to 0.015625%, the cytotoxicity, apoptosis-inducing effect and pro-apoptotic pathway was investigated, respectively. Our results showed that carteolol at concentrations above 0.03125% induced time- and dose-dependent growth retardation, cytopathic morphological changes and viability decline of HCEP cells. Moreover, carteolol induced G1 phase arrest, plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCEP cells. Furthermore, carteolol also induced activation of caspase-9 and -3, disruption of mitochondrial transmembrane potential, up-regulation the cytoplasmic amount of cytochrome c and apoptosis-inducing factor, and up-regulation of pro-apoptotic Bax and Bad, down-regulation of anti-apoptotic Bcl-2 and Bcl-xL. In conclusion, carteolol above 1/64 of its clinical therapeutic dosage has a time- and dose-dependent cytotoxicity to HCEP cells, which is achieved by inducing apoptosis via triggering Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway. PMID:27216471

  18. Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism

    PubMed Central

    Cao, Yudan; Cheng, Fangfang; Yao, Weifeng; Bao, Beihua; Zhang, Kaicheng; Zhang, Li; Ding, Anwei

    2016-01-01

    Pekinenin C is a casbane diterpenoid separated from the root of the traditional Chinese medicine, Euphorbia pekinensis Rupr., which is used as drug for the treatment of edema, ascites, and hydrothorax. Whereas pekinenin C exhibits severe cytotoxicity, the exact toxicity mechanism is unclear. In this study, the effects of pekinenin C on cell inhibition, cell cycle, and cell apoptosis were examined to explain its toxic mechanism. The proliferation of IEC-6 cells was accessed via MTT colorimetric assay after incubated with different concentrations of pekinenin C. Pekinenin C-treated IEC-6 cells labeled with RNase/PI and Annexin V/PI were analyzed by flow cytometric analyses for evaluation of cell cycle distribution and cell apoptosis, respectively. The apoptosis mechanism of pekinenin C on IEC-6 was investigated through assaying the activities of caspase-3, 8, 9 by enzyme-linked immunosorbent assay (ELISA), protein expression of Bax, Bcl-2, apoptosis-inducing factor (AIF), Apaf-1, Fas-associated death domain (FADD) and type 1-associated death domain (TRADD) by Western-blot, mRNA expression of Fas receptor (FasR), Fas ligand (FasL), tumor necrosis factor receptor (TNFR1) and NF-κB by RT-PCR. The results showed that pekinenin C has exhibited obvious IEC-6 cells toxicity and the IC50 value was 2.1 μg·mL−1. Typical apoptosis characteristics were observed under a transmission electron microscopy, and it was found that pekinenin C could cause G0/G1 phase arrest in IEC-6 cells in a dose-dependent manner and induce apoptosis of IEC-6 cells. Additionally, pekinenin C could increase the expressions of Bax, AIF, Apaf-1, FasR, FasL, TNFR1 and NF-κB, suppress the expression of Bcl-2, FADD and TRADD, then activate caspase-3, 8, 9 cascades, and at last result in apoptosis. These results demonstrated that pekinenin C effectively promoted cell apoptosis, and induced IEC-6 cells apoptosis through both the mitochondrial and death receptor pathways. PMID:27271594

  19. Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism.

    PubMed

    Cao, Yudan; Cheng, Fangfang; Yao, Weifeng; Bao, Beihua; Zhang, Kaicheng; Zhang, Li; Ding, Anwei

    2016-01-01

    Pekinenin C is a casbane diterpenoid separated from the root of the traditional Chinese medicine, Euphorbia pekinensis Rupr., which is used as drug for the treatment of edema, ascites, and hydrothorax. Whereas pekinenin C exhibits severe cytotoxicity, the exact toxicity mechanism is unclear. In this study, the effects of pekinenin C on cell inhibition, cell cycle, and cell apoptosis were examined to explain its toxic mechanism. The proliferation of IEC-6 cells was accessed via MTT colorimetric assay after incubated with different concentrations of pekinenin C. Pekinenin C-treated IEC-6 cells labeled with RNase/PI and Annexin V/PI were analyzed by flow cytometric analyses for evaluation of cell cycle distribution and cell apoptosis, respectively. The apoptosis mechanism of pekinenin C on IEC-6 was investigated through assaying the activities of caspase-3, 8, 9 by enzyme-linked immunosorbent assay (ELISA), protein expression of Bax, Bcl-2, apoptosis-inducing factor (AIF), Apaf-1, Fas-associated death domain (FADD) and type 1-associated death domain (TRADD) by Western-blot, mRNA expression of Fas receptor (FasR), Fas ligand (FasL), tumor necrosis factor receptor (TNFR1) and NF-κB by RT-PCR. The results showed that pekinenin C has exhibited obvious IEC-6 cells toxicity and the IC50 value was 2.1 μg·mL(-1). Typical apoptosis characteristics were observed under a transmission electron microscopy, and it was found that pekinenin C could cause G0/G1 phase arrest in IEC-6 cells in a dose-dependent manner and induce apoptosis of IEC-6 cells. Additionally, pekinenin C could increase the expressions of Bax, AIF, Apaf-1, FasR, FasL, TNFR1 and NF-κB, suppress the expression of Bcl-2, FADD and TRADD, then activate caspase-3, 8, 9 cascades, and at last result in apoptosis. These results demonstrated that pekinenin C effectively promoted cell apoptosis, and induced IEC-6 cells apoptosis through both the mitochondrial and death receptor pathways. PMID:27271594

  20. Polarization of the epithelial layer and apical localization of integrins are required for engulfment of apoptotic cells in the Drosophila ovary

    PubMed Central

    Meehan, Tracy L.; Kleinsorge, Sarah E.; Timmons, Allison K.; Taylor, Jeffrey D.; McCall, Kimberly

    2015-01-01

    ABSTRACT Inefficient clearance of dead cells or debris by epithelial cells can lead to or exacerbate debilitating conditions such as retinitis pigmentosa, macular degeneration, chronic obstructive pulmonary disease and asthma. Despite the importance of engulfment by epithelial cells, little is known about the molecular changes that are required within these cells. The misregulation of integrins has previously been associated with disease states, suggesting that a better understanding of the regulation of receptor trafficking could be key to treating diseases caused by defects in phagocytosis. Here, we demonstrate that the integrin heterodimer αPS3/βPS becomes apically enriched and is required for engulfment by the epithelial follicle cells of the Drosophila ovary. We found that integrin heterodimer localization and function is largely directed by the α-subunit. Moreover, proper cell polarity promotes asymmetric integrin enrichment, suggesting that αPS3/βPS trafficking occurs in a polarized fashion. We show that several genes previously known for their roles in trafficking and cell migration are also required for engulfment. Moreover, as in mammals, the same α-integrin subunit is required by professional and non-professional phagocytes and migrating cells in Drosophila. Our findings suggest that migrating and engulfing cells use common machinery, and demonstrate a crucial role for integrin function and polarized trafficking of integrin subunits during engulfment. This study also establishes the epithelial follicle cells of the Drosophila ovary as a powerful model for understanding the molecular changes required for engulfment by a polarized epithelium. PMID:26398951

  1. Apoptotic Cell Death in Neuroblastoma

    PubMed Central

    Li, Yuanyuan; Nakagawara, Akira

    2013-01-01

    Neuroblastoma (NB) is one of the most common malignant solid tumors in childhood, which derives from the sympathoadrenal lineage of the neural crest and exhibits extremely heterogeneous biological and clinical behaviors. The infant patients frequently undergo spontaneous regression even with metastatic disease, whereas the patients of more than one year of age who suffer from disseminated disease have a poor outcome despite intensive multimodal treatment. Spontaneous regression in favorable NBs has been proposed to be triggered by nerve growth factor (NGF) deficiency in the tumor with NGF dependency for survival, while aggressive NBs have defective apoptotic machinery which enables the tumor cells to evade apoptosis and confers the resistance to treatment. This paper reviews the molecules and pathways that have been recently identified to be involved in apoptotic cell death in NB and discusses their potential prospects for developing more effective therapeutic strategies against aggressive NB. PMID:24709709

  2. The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity.

    PubMed

    Polgar, Noemi; Lee, Amanda J; Lui, Vanessa H; Napoli, Josephine A; Fogelgren, Ben

    2015-08-01

    The highly conserved exocyst protein complex regulates polarized exocytosis of subsets of secretory vesicles. A previous study reported that shRNA knockdown of an exocyst central subunit, Sec10 (Sec10-KD) in Madin-Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. We used three-dimensional collagen cultures of MDCK cells to further investigate the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis. Sec10-KD cysts initially demonstrated undisturbed lumen formation although later displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, control cells maintained normal homeostasis, while Sec10-KD cysts displayed numerous apoptotic cells extruded basally into the collagen matrix. Sec10-KD MDCK cells were also more sensitive to apoptotic triggers than controls. These phenotypes were reversed by restoring Sec10 expression with shRNA-resistant human Sec10. Apico-basal polarity appeared normal in Sec10-KD cysts, whereas mitotic spindle angles differed significantly from controls, suggesting a planar cell polarity defect. In addition, analysis of renal tubules in a newly generated kidney-specific Sec10-knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules; abnormal epithelial cell extrusion was also observed, supporting our in vitro results. We hypothesize that, in Sec10-KD cells, the disrupted exocyst activity results in increased apoptotic sensitivity through defective primary cilia signaling and that, in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis. PMID:26040895

  3. Induction of apoptosis in oral epithelial cells by Candida albicans.

    PubMed

    Villar, C Cunha; Chukwuedum Aniemeke, J; Zhao, X-R; Huynh-Ba, G

    2012-12-01

    During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C. albicans-induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid-late apoptotic alterations. At the molecular level, C. albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase-3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic-like fragments. Finally, five anti-apoptotic genes were significantly upregulated and two pro-apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C. albicans, but fail to progress and promote apoptotic cell death. PMID:23134609

  4. Stabilization of apoptotic cells: generation of zombie cells.

    PubMed

    Oropesa-Ávila, M; Andrade-Talavera, Y; Garrido-Maraver, J; Cordero, M D; de la Mata, M; Cotán, D; Paz, M V; Pavón, A D; Alcocer-Gómez, E; de Lavera, I; Lema, R; Zaderenko, A P; Rodríguez-Moreno, A; Sánchez-Alcázar, J A

    2014-01-01

    Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn(2+) (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy. PMID:25118929

  5. Stabilization of apoptotic cells: generation of zombie cells

    PubMed Central

    Oropesa-Ávila, M; Andrade-Talavera, Y; Garrido-Maraver, J; Cordero, M D; de la Mata, M; Cotán, D; Paz, M V; Pavón, A D; Alcocer-Gómez, E; de Lavera, I; Lema, R; Zaderenko, A P; Rodríguez-Moreno, A; Sánchez-Alcázar, J A

    2014-01-01

    Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn2+ (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy. PMID:25118929

  6. Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses.

    PubMed

    Cunin, P; Beauvillain, C; Miot, C; Augusto, J-F; Preisser, L; Blanchard, S; Pignon, P; Scotet, M; Garo, E; Fremaux, I; Chevailler, A; Subra, J-F; Blanco, P; Wilson, M R; Jeannin, P; Delneste, Y

    2016-01-01

    Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu(-/-) mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases. PMID:27148688

  7. Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses

    PubMed Central

    Cunin, P; Beauvillain, C; Miot, C; Augusto, J-F; Preisser, L; Blanchard, S; Pignon, P; Scotet, M; Garo, E; Fremaux, I; Chevailler, A; Subra, J-F; Blanco, P; Wilson, M R; Jeannin, P; Delneste, Y

    2016-01-01

    Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu−/− mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases. PMID:27148688

  8. The Dynamics of Apoptotic Cell Clearance.

    PubMed

    Elliott, Michael R; Ravichandran, Kodi S

    2016-07-25

    The phagocytic clearance of dying cells in a tissue is a highly orchestrated series of intercellular events coordinated by a complex signaling network. Recent data from genetic, biochemical, and live-imaging approaches have greatly enhanced our understanding of the dynamics of cell clearance and how the process is orchestrated at the cellular and tissue levels. We discuss how networks regulating apoptotic cell clearance are integrated to enable a rapid, efficient, and high-capacity clearance system within tissues. PMID:27459067

  9. Conjugated Polyelectrolyte Nanoparticles for Apoptotic Cell Imaging.

    PubMed

    Liu, Yu; Wu, Pan; Jiang, Jianhua; Wu, Jiatao; Chen, Yan; Tan, Ying; Tan, Chunyan; Jiang, Yuyang

    2016-08-31

    Three anionic conjugated polyelectrolytes (CPEs) with poly(p-phenylene ethynylene thiophene) backbones were designed and synthesized, among which PPET3-CO2Na showed greater molar extinction coefficient with red-shifted bands in both absorption and emission spectra compared to the well-studied PPE-CO2Na polymer. PPET3-CO2Na was thus chosen to construct CPE-based nanoparticles (CPNs) with cationic octaarginine (R8) peptide through electrostatic-interaction-induced self-assembly. Due to plasma membrane permeabilization and mitochondrial outer membrane permeabilization (MOMP) in early apoptotic cells, PPET3/R8 CPNs demonstrated excellent colocalization with MitoTracker Red in apoptotic cells instead of normal cells, which had potential application in cell imaging for early apoptosis recognition. PMID:27525500

  10. Human bronchial epithelial cells exposed in vitro to diesel exhaust particles exhibit alterations in cell rheology and cytotoxicity associated with decrease in antioxidant defenses and imbalance in pro- and anti-apoptotic gene expression.

    PubMed

    Seriani, Robson; de Souza, Claudia Emanuele Carvalho; Krempel, Paloma Gava; Frias, Daniela Perroni; Matsuda, Monique; Correia, Aristides Tadeu; Ferreira, Márcia Zotti Justo; Alencar, Adriano Mesquita; Negri, Elnara Marcia; Saldiva, Paulo Hilário Nascimento; Mauad, Thais; Macchione, Mariangela

    2016-05-01

    Diesel exhaust particles (DEPs) from diesel engines produce adverse alterations in cells of the airways by activating intracellular signaling pathways and apoptotic gene overexpression, and also by influencing metabolism and cytoskeleton changes. This study used human bronchial epithelium cells (BEAS-2B) in culture and evaluates their exposure to DEPs (15ug/mL for 1 and 2 h) in order to determine changes to cell rheology (viscoelasticity) and gene expression of the enzymes involved in oxidative stress, apoptosis, and cytotoxicity. BEAS-2B cells exposed to DEPs were found to have a significant loss in stiffness, membrane stability, and mitochondrial activity. The genes involved in apoptosis [B cell lymphoma 2 (BCL-2 and caspase-3)] presented inversely proportional expressions (p = 0.05, p = 0.01, respectively), low expression of the genes involved in antioxidant responses [SOD1 (superoxide dismutase 1); SOD2 (superoxide dismutase 2), and GPx (glutathione peroxidase) (p = 0.01)], along with an increase in cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) (p = 0.01). These results suggest that alterations in cell rheology and cytotoxicity could be associated with oxidative stress and imbalance between pro- and anti-apoptotic genes. PMID:26856867

  11. Epithelial Cell Adhesion Molecule

    PubMed Central

    Trzpis, Monika; McLaughlin, Pamela M.J.; de Leij, Lou M.F.H.; Harmsen, Martin C.

    2007-01-01

    The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of ∼40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis. PMID:17600130

  12. Apoptotic cell death induced by intracellular proteolysis.

    PubMed

    Williams, M S; Henkart, P A

    1994-11-01

    To mimic the injection of granzymes into target cells by cytotoxic lymphocytes or the activation of endogenous proteases in programmed cell death, the proteases chymotrypsin, proteinase K, or trypsin were loaded into the cytoplasm of several different cell types using the osmotic lysis of pinosomes technique. Internalization of these proteases caused cell lysis within several hours, accompanied by extensive nuclear damage in most but not all combinations of target cells and proteases. This nuclear damage, quantitated by DNA release from nuclei, was associated with apoptotic features including DNA fragmentation into nucleosomal ladders, chromatin condensation, nuclear fragmentation, and membrane blebbing. Agents reported to block programmed cell death, including aurintricarboxylic acid, inhibitors of energy metabolism, and protein or RNA synthesis, failed to block this protease-induced death, although some inhibited nuclear damage. In separate experiments, introduction of staphylococcal nuclease into cells led to near complete (at least 75% of total) nucleosomal DNA fragmentation within 6 to 8 h. Condensation of chromatin did not accompany this fragmentation to the same extent, and there was approximately a 10-h lag between half-maximal DNA fragmentation and 50% loss of membrane integrity. The results suggest that activation of intracellular proteases during cell death by any molecular pathway could give rise to apoptotic morphology and DNA fragmentation. PMID:7930626

  13. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

    PubMed

    Kajita, Mihoko; Fujita, Yasuyuki

    2015-07-01

    During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; 'loser' cells are eliminated from tissues and consequently 'winner' cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells show more variable phenotypes in mammals, such as cell death-independent apical extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighbouring transformed cells via cytoskeletal proteins by the process named epithelial defence against cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals. PMID:25991731

  14. Epithelial stem cells.

    PubMed

    Draheim, Kyle M; Lyle, Stephen

    2011-01-01

    It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process. PMID:21618097

  15. Apoptotic cell clearance: basic biology and therapeutic potential

    PubMed Central

    Poon, Ivan K. H.; Lucas, Christopher D.

    2014-01-01

    Prompt removal of apoptotic cells by phagocytes is important for maintaining tissue homeostasis. The molecular and cellular events that underpin apoptotic cell recognition and uptake, and the subsequent biological responses are increasingly better defined. The detection and disposal of apoptotic cells generally promote an anti-inflammatory response at the tissue level, as well as immunological tolerance. Consequently, defects in apoptotic cell clearance have been linked with a variety of inflammatory diseases and autoimmunity. Conversely, under certain conditions such as killing tumour cells by specific cell death inducers, the recognition of apoptotic tumour cells can promote an immunogenic response and anti-tumour immunity. Here, we review the current understanding of the complex process of apoptotic cell clearance in physiology and pathology, and discuss how this knowledge could be harnessed for new therapeutic strategies. PMID:24481336

  16. The role of airway macrophages in apoptotic cell clearance following acute and chronic lung inflammation.

    PubMed

    Grabiec, Aleksander M; Hussell, Tracy

    2016-07-01

    Acute and chronic inflammatory responses in the lung are associated with the accumulation of large quantities of immune and structural cells undergoing apoptosis, which need to be engulfed by phagocytes in a process called 'efferocytosis'. Apoptotic cell recognition and removal from the lung is mediated predominantly by airway macrophages, though immature dendritic cells and non-professional phagocytes, such as epithelial cells and mesenchymal cells, can also display this function. Efficient clearance of apoptotic cells from the airways is essential for successful resolution of inflammation and the return to lung homeostasis. Disruption of this process leads to secondary necrosis of accumulating apoptotic cells, release of necrotic cell debris and subsequent uncontrolled inflammatory activation of the innate immune system by the released 'damage associated molecular patterns' (DAMPS). To control the duration of the immune response and prevent autoimmune reactions, anti-inflammatory signalling cascades are initiated in the phagocyte upon apoptotic cell uptake, mediated by a range of receptors that recognise specific phospholipids or proteins externalised on, or secreted by, the apoptotic cell. However, prolonged activation of apoptotic cell recognition receptors, such as the family of receptor tyrosine kinases Tyro3, Axl and MerTK (TAM), may delay or prevent inflammatory responses to subsequent infections. In this review, we will discuss recent advances in our understanding of the mechanism controlling apoptotic cell recognition and removal from the lung in homeostasis and during inflammation, the contribution of defective efferocytosis to chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease, asthma and cystic fibrosis, and implications of the signals triggered by apoptotic cells in the susceptibility to pulmonary microbial infections. PMID:26957481

  17. Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages.

    PubMed

    Petrusca, Daniela N; Gu, Yuan; Adamowicz, Jeremy J; Rush, Natalia I; Hubbard, Walter C; Smith, Patricia A; Berdyshev, Evgeni V; Birukov, Konstantin G; Lee, Chao-Hung; Tuder, Rubin M; Twigg, Homer L; Vandivier, R William; Petrache, Irina

    2010-12-17

    A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM. PMID:20956540

  18. Apoptotic Cells Induce NF-κB and Inflammasome Negative Signaling

    PubMed Central

    Grau, Amir; Tabib, Adi; Grau, Inna; Reiner, Inna; Mevorach, Dror

    2015-01-01

    As they undergo phagocytosis, most early apoptotic cells negatively regulate proinflammatory signaling and were suggested as a major mechanism in the resolution of inflammation. The dextran sulfate sodium model is generally viewed as an epithelial damage model suited to investigate innate immune responses. Macrophages primed with LPS and subsequently exposed to DSS secrete high levels of IL-1β in an NLRP3-, ASC-, and caspase-1-dependent manner. The aim of this research was to test the therapeutic effect of a single dose of apoptotic cells in a DSS-colitis model and to explore possible mechanisms. Primary peritoneal macrophages, the DSS mice model, and Nlrp3-deficient mice, were used to assess the effect apoptotic cells on colitis. Immunohistochemistry, flow-cytometer, and western blots helped to explore the effect and mechanisms. Using a variety of NLRP3 triggering mechanisms, we show that apoptotic cells negatively regulate NF-κB and NLRP3 activation in primary peritoneal macrophages, at pre- and post-transcription levels, via inhibition of reactive oxygen species, lysosomal stabilization, and blocking K+ efflux. This property of apoptotic cells is demonstrated in a dramatic clinical, histological, and immunological amelioration of DSS colitis in Balb/c and B6 mice following a single administration of apoptotic cells. PMID:25822487

  19. Apoptotic cells selectively uptake minor glycoforms of vitronectin from serum.

    PubMed

    Malagolini, Nadia; Catera, Mariangela; Osorio, Hugo; Reis, Celso A; Chiricolo, Mariella; Dall'Olio, Fabio

    2013-04-01

    Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the α2,6-sialyl-specific lectin from Sambucus nigra agglutinin (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few α2,6-linked sialic acid residues. Despite their poor α2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the α2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-β2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the α2,6-sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis. PMID:23381642

  20. Apoptotic Cells Initiate Endothelial Cell Sprouting via Electrostatic Signaling

    PubMed Central

    Weihua, Zhang; Tsan, Rachel; Schroit, Alan J.; Fidler, Isaiah J.

    2006-01-01

    Angiogenesis, the development of new blood vessels from preexisting vessels, is crucial to tissue growth, repair, and maintenance. This process begins with the formation of endothelial cell sprouts followed by the proliferation and migration of neighboring endothelial cells along the pre-formed extensions. The initiating event and mechanism of sprouting is not known. We demonstrate that the phenotypic expression of negative-charged membrane surface in apoptotic cells initiates the formation of directional endothelial cell sprouts that extend toward the dying cells by a mechanism that involves endothelial cell membrane hyperpolarization and cytoskeleton reorganization but is independent of diffusible molecules. PMID:16357162

  1. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  2. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

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

  3. In vitro study of immunosuppressive effect of apoptotic cells*

    PubMed Central

    Zhang, Wen-jin; Zheng, Shu-sen

    2005-01-01

    Recent studies revealed that apoptotic cells are actively involved in immunosuppression and anti-inflammation. After being phagocytosed by macrophages, apoptotic cells can actively regulate cytokines secretion from lipopolysaccharide (LPS)-stimulated macrophages, in which the secretion of immunosuppressive cytokines such as interleukin-10 (IL-10) is increased while the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), interleukin-1beta (IL-1β) and leukin-8 (IL-8) are suppressed. In this paper, we first present evidence that phagocytosed apoptotic cells regulate cytokine secretion of LPS-stimulated macrophages, but also inhibit the activation of T lymphocytes stimulated by ConA. These data suggest that apoptotic cells can alter the biological behavior of macrophages which gain immunosuppressive property. PMID:16130196

  4. Surface code—biophysical signals for apoptotic cell clearance

    NASA Astrophysics Data System (ADS)

    Biermann, Mona; Maueröder, Christian; Brauner, Jan M.; Chaurio, Ricardo; Janko, Christina; Herrmann, Martin; Muñoz, Luis E.

    2013-12-01

    Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

  5. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

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

  6. Apoptotic cell death in rat epididymis following epichlorohydrin treatment.

    PubMed

    Lee, I-C; Kim, K-H; Kim, S-H; Baek, H-S; Moon, C; Kim, S-H; Yun, W-K; Nam, K-H; Kim, H-C; Kim, J-C

    2013-06-01

    Epichlorohydrin (ECH) is an antifertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility. This study identified the time course of apoptotic cell death in rat epididymides after ECH treatment. Rats were administrated with a single oral dose of ECH (50 mg/kg). ECH-induced apoptotic changes were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and its related mechanism was confirmed by Western blot analysis and colorimetric assay. The TUNEL assay showed that the number of apoptotic cells increased at 8 h, reached a maximum level at 12 h, and then decreased progressively. The Western blot analysis demonstrated no significant changes in proapoptotic Bcl-2-associated X (Bax) and anti-apoptotic Bcl-2 expression during the time course of the study. However, phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and phospho-c-Jun amino-terminal kinase (p-JNK) expression increased at 8-24 h. Caspase-3 and caspase-8 activities also increased at 8-48 h and 12-48 h, respectively, in the same manner as p-p38 MAPK and p-JNK expression. These results indicate that ECH induced apoptotic changes in rat epididymides and that the apoptotic cell death may be related more to the MAPK pathway than to the mitochondrial pathway. PMID:23386780

  7. Monitoring circulating apoptotic cells by in-vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Wei, Xunbin; Tan, Yuan; Chen, Yun; Zhang, Li; Li, Yan; Liu, Guangda; Wu, Bin; Wang, Chen

    2008-02-01

    Chemotherapies currently constitute one main venue of cancer treatment. For a large number of adult and elderly patients, however, treatment options are poor. These patients may suffer from disease that is resistant to conventional chemotherapy or may not be candidates for curative therapies because of advanced age or poor medical conditions. To control disease in these patients, new therapies must be developed that are selectively targeted to unique characteristics of tumor cell growth and metastasis. A reliable early evaluation and prediction of response to the chemotherapy is critical to its success. Chemotherapies induce apoptosis in tumor cells and a portion of such apoptotic cancer cells may be present in the circulation. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method that enables real-time detection and quantification of circulating cells without blood extraction. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 minutes. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention.

  8. Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells

    NASA Astrophysics Data System (ADS)

    Roy, Prathik; Periasamy, Arun Prakash; Lin, Chiu-Ya; Her, Guor-Mour; Chiu, Wei-Jane; Li, Chi-Lin; Shu, Chia-Lun; Huang, Chih-Ching; Liang, Chi-Te; Chang, Huan-Tsung

    2015-01-01

    Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications.Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with

  9. Engulfment of apoptotic cells: signals for a good meal.

    PubMed

    Ravichandran, Kodi S; Lorenz, Ulrike

    2007-12-01

    The clearance of apoptotic cells by phagocytes is an integral component of normal life, and defects in this process can have significant implications for self tolerance and autoimmunity. Recent studies have provided new insights into the engulfment process, including how phagocytes seek apoptotic cells, how they recognize and ingest these targets and how they maintain cellular homeostasis after the 'meal'. Several new factors that regulate engulfment have been identified, whereas the roles of some of the older players require revision. This Review focuses on these recent developments and attempts to highlight some of the important questions in this field. PMID:18037898

  10. Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization

    PubMed Central

    Fornetti, J; Flanders, K C; Henson, P M; Tan, A-C; Borges, V F; Schedin, P

    2016-01-01

    After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and

  11. Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization.

    PubMed

    Fornetti, J; Flanders, K C; Henson, P M; Tan, A-C; Borges, V F; Schedin, P

    2016-02-01

    After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and

  12. Identification and apoptotic potential of T-2 toxin metabolites in human cells.

    PubMed

    Weidner, Maria; Welsch, Tanja; Hübner, Florian; Schwerdt, Gerald; Gekle, Michael; Humpf, Hans-Ulrich

    2012-06-01

    The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell culture studies using cells of human origin represent a potent model system to study the metabolic fate of T-2 toxin as well as the cytotoxicity in vitro. In this study the metabolism of T-2 toxin was analyzed in a cell line derived from human colon carcinoma cells (HT-29) and primary human renal proximal tubule epithelial cells (RPTEC) using high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (HPLC-FTMS). Both cell types metabolized T-2 toxin to a variety of compounds. Furthermore, cell cycle analysis in RPTEC proved the apoptotic effect of T-2 toxin and its metabolites HT-2 toxin and neosolaniol in micromolar concentrations. PMID:22551244

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

    PubMed Central

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

    2016-01-01

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

  14. PDT-treated apoptotic cells induce macrophage synthesis NO

    NASA Astrophysics Data System (ADS)

    Song, S.; Xing, D.; Zhou, F. F.; Chen, W. R.

    2009-11-01

    Nitric oxide (NO) is a biologically active molecule which has multi-functional in different species. As a second messenger and neurotransmitter, NO is not only an important regulatory factor between cells' information transmission, but also an important messenger in cell-mediated immunity and cytotoxicity. On the other side, NO is involving in some diseases' pathological process. In pathological conditions, the macrophages are activated to produce a large quantity of nitric oxide synthase (iNOS), which can use L-arginine to produce an excessive amount of NO, thereby killing bacteria, viruses, parasites, fungi, tumor cells, as well as in other series of the immune process. In this paper, photofrin-based photodynamic therapy (PDT) was used to treat EMT6 mammary tumors in vitro to induce apoptotic cells, and then co-incubation both apoptotic cells and macrophages, which could activate macrophage to induce a series of cytotoxic factors, especially NO. This, in turn, utilizes macrophages to activate a cytotoxic response towards neighboring tumor cells. These results provided a new idea for us to further study the immunological mechanism involved in damaging effects of PDT, also revealed the important function of the immune effect of apoptotic cells in PDT.

  15. Apoptotic cell signaling in cancer progression and therapy.

    PubMed

    Plati, Jessica; Bucur, Octavian; Khosravi-Far, Roya

    2011-04-01

    Apoptosis is a tightly regulated cell suicide program that plays an essential role in the development and maintenance of tissue homeostasis by eliminating unnecessary or harmful cells. Impairment of this native defense mechanism promotes aberrant cellular proliferation and the accumulation of genetic defects, ultimately resulting in tumorigenesis, and frequently confers drug resistance to cancer cells. The regulation of apoptosis at several levels is essential to maintain the delicate balance between cellular survival and death signaling that is required to prevent disease. Complex networks of signaling pathways act to promote or inhibit apoptosis in response to various cues. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Various upstream signaling pathways can modulate apoptosis by converging on, and thereby altering the activity of, common central control points within the apoptotic signaling pathways, which involve the BCL-2 family proteins, inhibitor of apoptosis (IAP) proteins, and FLICE-inhibitory protein (c-FLIP). This review highlights the role of these fundamental regulators of apoptosis in the context of both normal apoptotic signaling mechanisms and dysregulated apoptotic pathways that can render cancer cells resistant to cell death. In addition, therapeutic strategies aimed at modulating the activity of BCL-2 family proteins, IAPs, and c-FLIP for the targeted induction of apoptosis are briefly discussed. PMID:21340093

  16. ELMO1 signaling in apoptotic germ cell clearance and spermatogenesis.

    PubMed

    Elliott, Michael R; Ravichandran, Kodi S

    2010-10-01

    Apoptosis and the subsequent removal of dying cells are crucial processes for tissue development and maintenance. Although we are beginning to understand the signaling pathways that control the phagocytic clearance of apoptotic cells, the physiological relevance of these pathways is lacking. During spermatogenesis, over half of the developing germ cells eventually die by apoptosis, yet the signaling pathways that regulate the phagocytic clearance of these dying cells or the impact of this clearance on development and maintenance of the germ cell population is not well understood. The ELMO1/Dock180 proteins form an evolutionarily conserved signaling module that functions as a bipartite guanine nucleotide exchange factor for the small GTPase Rac. The subsequent Rac-dependent cytoskeletal changes play an important role in the physical engulfment of apoptotic cells. Recent findings demonstrate an in vivo role for ELMO1-dependent clearance in the testes, with implications for spermatogenesis. Here we will discuss the role of apoptotic cell clearance during spermatogenesis, with a particular emphasis on ELMO1/Dock180 signaling. PMID:20958313

  17. Emerging roles for lipids in non-apoptotic cell death.

    PubMed

    Magtanong, L; Ko, P J; Dixon, S J

    2016-07-01

    Non-apoptotic regulated cell death (RCD) is essential to maintain organismal homeostasis and may be aberrantly activated during certain pathological states. Lipids are emerging as key components of several non-apoptotic RCD pathways. For example, a direct interaction between membrane phospholipids and the pore-forming protein mixed lineage kinase domain-like (MLKL) is needed for the execution of necroptosis, while the oxidative destruction of membrane polyunsaturated fatty acids (PUFAs), following the inactivation of glutathione peroxidase 4 (GPX4), is a requisite gateway to ferroptosis. Here, we review the roles of lipids in the initiation and execution of these and other forms of non-apoptotic cell death. We also consider new technologies that are allowing for the roles of lipids and lipid metabolism in RCD to be probed in increasingly sophisticated ways. In certain cases, this new knowledge may enable the development of therapies that target lipids and lipid metabolic processes to enhance or suppress specific non-apoptotic RCD pathways. PMID:26967968

  18. Retinoic acid promotes primary fetal alveolar epithelial type II cell proliferation and differentiation to alveolar epithelial type I cells.

    PubMed

    Gao, Rui-wei; Kong, Xiang-yong; Zhu, Xiao-xi; Zhu, Guo-qing; Ma, Jin-shuai; Liu, Xiu-xiang

    2015-05-01

    Retinoic acid (RA) plays an important role in lung development and maturation. Many stimuli can induce alveolar epithelial cell damage which will result in the injury of lung parenchyma. The aim of this study was to observe the effect of RA on the proliferation and differentiation of primary fetal alveolar epithelial type II cells (fAECIIs). Primary fAECIIs were isolated from fetal rats at 19 d of gestation and purified by a differential centrifugation and adhesion method. The cells were randomly divided into control (dimethyl sulfoxide, DMSO) and RA groups. Cell proliferation, viability, apoptosis, cycle, and expression of target protein were examined at 24, 48, and 72 h. We found that the proliferation and viability of cells in the RA-exposed group significantly increased compared with the DMSO control group. The proportion (%) of cells in the G2 and S phases in the RA group was significantly higher than that in control group cells. The proportion (%) of both early apoptotic cells and late apoptotic cells decreased significantly in cells exposed to RA compared with cells exposed to DMSO. RA significantly enhanced the expression of aquaporin 5 (AQP5). The expression level of pulmonary surfactant C (SPC) was elevated after cells were exposed to RA for 24 and 72 h but was inhibited when cells were exposed to RA for 48 h. These results suggest that RA promotes fAECII proliferation by improving cell viability, promoting S phase entry and inhibiting apoptosis and RA promotes fAECIIs differentiation to alveolar epithelial type I cells (AECIs). PMID:25515249

  19. Sp3 regulates fas expression in lung epithelial cells.

    PubMed Central

    Pang, H; Miranda, K; Fine, A

    1998-01-01

    By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5' flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter-luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60-70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells. PMID:9639581

  20. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

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

  1. IGF-1 protects tubular epithelial cells during injury via activation of ERK/MAPK signaling pathway

    PubMed Central

    Wu, Zengbin; Yu, Yang; Niu, Lei; Fei, Aihua; Pan, Shuming

    2016-01-01

    Injury of renal tubular epithelial cells can induce acute renal failure and obstructive nephropathy. Previous studies have shown that administration of insulin-like growth factor-1 (IGF-1) ameliorates the renal injury in a mouse unilateral ureteral obstruction (UUO) model, whereas the underlying mechanisms are not completely understood. Here, we addressed this question. We found that the administration of IGF-1 significantly reduced the severity of the renal fibrosis in UUO. By analyzing purified renal epithelial cells, we found that IGF-1 significantly reduced the apoptotic cell death of renal epithelial cells, seemingly through upregulation of anti-apoptotic protein Bcl-2, at protein but not mRNA level. Bioinformatics analyses and luciferase-reporter assay showed that miR-429 targeted the 3′-UTR of Bcl-2 mRNA to inhibit its protein translation in renal epithelial cells. Moreover, IGF-1 suppressed miR-429 to increase Bcl-2 in renal epithelial cells to improve survival after UUO. Furthermore, inhibition of ERK/MAPK signaling pathway in renal epithelial cells abolished the suppressive effects of IGF-1 on miR-429 activation, and then the enhanced effects on Bcl-2 in UUO. Thus, our data suggest that IGF-1 may protect renal tubular epithelial cells via activation of ERK/MAPK signaling pathway during renal injury. PMID:27301852

  2. Cyclooxygenase-2-dependent phosphorylation of the pro-apoptotic protein Bad inhibits tonicity-induced apoptosis in renal medullary cells.

    PubMed

    Küper, Christoph; Bartels, Helmut; Beck, Franz-X; Neuhofer, Wolfgang

    2011-11-01

    During antidiuresis, cell survival in the renal medulla requires cyclooxygenase-2 (COX-2) activity. We have recently found that prostaglandin E2 (PGE2) promotes cell survival by phosphorylation and, hence, inactivation of the pro-apoptotic protein Bad during hypertonic stress in Madin-Darby canine kidney (MDCK) cells in vitro. Here we determine the role of COX-2-derived PGE(2) on phosphorylation of Bad and medullary apoptosis in vivo using COX-2-deficient mice. Both wild-type and COX-2-knockout mice constitutively expressed Bad in tubular epithelial cells of the renal medulla. Dehydration caused a robust increase in papillary COX-2 expression, PGE2 excretion, and Bad phosphorylation in wild-type, but not in the knockout mice. The abundance of cleaved caspase-3, a marker of apoptosis, was significantly higher in papillary homogenates, especially in tubular epithelial cells of the knockout mice. Knockdown of Bad in MDCK cells decreased tonicity-induced caspase-3 activation. Furthermore, the addition of PGE2 to cells with knockdown of Bad had no effect on caspase-3 activation; however, PGE2 caused phosphorylation of Bad and substantially improved cell survival in mock-transfected cells. Thus, tonicity-induced COX-2 expression and PGE2 synthesis in the renal medulla entails phosphorylation and inactivation of the pro-apoptotic protein Bad, thereby counteracting apoptosis in renal medullary epithelial cells. PMID:21716255

  3. Opium induces apoptosis in Jurkat cells via promotion of pro-apoptotic and inhibition of anti-apoptotic molecules

    PubMed Central

    Arababadi, Mohammad Kazemi; Asadikaram, Gholamreza

    2016-01-01

    Objective(s): The aim of this study was to determine the important molecules involved in apoptosis induction by opium in Jurkat cell line. Materials and Methods: Jurkat cells were incubated 48 hrs with 2.86×10-5 g/ml concentration of opium and apoptosis as well as expression levels of related molecules were measured. Results: Our results demonstrated that 50.3±0.2 percent of opium treated Jurkat cells were revealed apoptotic features. The levels of mRNA of several pro-apoptotic and anti-apoptotic molecules were increased and decreased, respectively, in the opium treated cells. The results also demonstrated that expression levels of BCL2, DFFA and NOL3 as anti-apoptotic molecules were increased in the opium treated cells. Conclusion: It seems that opium induces apoptosis in Jurkat cells via both intrinsic and extrinsic pathways. Although opium induces apoptosis in the cells but increased expression of some anti-apoptotic molecules may be a normal resistance of the cell for death. PMID:27081468

  4. Apoptotic Death of Cancer Stem Cells for Cancer Therapy

    PubMed Central

    He, Ying-Chun; Zhou, Fang-Liang; Shen, Yi; Liao, Duan-Fang; Cao, Deliang

    2014-01-01

    Cancer stem cells (CSCs) play crucial roles in tumor progression, chemo- and radiotherapy resistance, and recurrence. Recent studies on CSCs have advanced understanding of molecular oncology and development of novel therapeutic strategies. This review article updates the hypothesis and paradigm of CSCs with a focus on major signaling pathways and effectors that regulate CSC apoptosis. Selective CSC apoptotic inducers are introduced and their therapeutic potentials are discussed. These include synthetic and natural compounds, antibodies and recombinant proteins, and oligonucleotides. PMID:24823879

  5. Reduced association of anti-apoptotic protein Mcl-1 with E3 ligase Mule increases the stability of Mcl-1 in breast cancer cells

    PubMed Central

    Pervin, S; Tran, A; Tran, L; Urman, R; Braga, M; Chaudhuri, G; Singh, R

    2011-01-01

    Background: Mechanisms that increase resistance to apoptosis help promote cellular transformation. Cancer cells have deregulated apoptotic pathways, where increased expression and stability of anti-apoptotic proteins Mcl-1 and Bcl-2 increases resistance to apoptosis. Pathways that increase the stability of proteins in cancer cells remain poorly understood. Methods: Using human mammary epithelial and established breast cancer cell lines, we assessed the mechanisms that increase the stability of anti-apoptotic proteins in breast cancer cells by caspase assay, western blot, small-inhibitory RNA treatment and immunoprecipitation. Results: While breast cancer cells were resistant to de novo inhibition of protein synthesis, a rapid proteosome-mediated degradation of Mcl-1 and Bcl-2 induced apoptosis in mammary epithelial cells. Although Mule, an E3 ligase that targets Mcl-1 for degradation was expressed in mammary epithelial and breast cancer cell lines, rapid increase of polyubiquitinated Mcl-1 and Bcl-2 was detected only in mammary epithelial cells. Only transient formation of the Mule–Mcl-1 complex was detected in breast cancer cells. Downregulation of pERK1/2 in breast cancer cells reduced Mcl-1 levels and increased Mcl-1/Mule complex. Conclusion: Our findings suggest that reduced Mule/Mcl-1 complex has a significant role in increasing the stability of Mcl-1 in breast cancer cells and increased resistance to apoptosis. PMID:21730980

  6. Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells.

    PubMed

    Chen, Xiuping; Guo, Feiye; LeBlanc, Michelle E; Ding, Ying; Zhang, Chenming; Shakya, Akhalesh; Li, Wei

    2016-08-01

    Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance. PMID:27184668

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

    PubMed Central

    Fehrenbach, Heinz

    2001-01-01

    In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2) cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today. PMID:11686863

  8. Epithelial organization, cell polarity and tumorigenesis.

    PubMed

    McCaffrey, Luke Martin; Macara, Ian G

    2011-12-01

    Epithelial cells comprise the foundation for the majority of organs in the mammalian body, and are the source of approximately 90% of all human cancers. Characteristically, epithelial cells form intercellular adhesions, exhibit apical/basal polarity, and orient their mitotic spindles in the plane of the epithelial sheet. Defects in these attributes result in the tissue disorganization associated with cancer. Epithelia undergo self-renewal from stem cells, which might in some cases be the cell of origin for cancers. The PAR polarity proteins are master regulators of epithelial organization, and are closely linked to signaling pathways such as Hippo, which orchestrate proliferation and apoptosis to control organ size. 3D ex vivo culture systems can now faithfully recapitulate epithelial organ morphogenesis, providing a powerful approach to study both normal development and the initiating events in carcinogenesis. PMID:21782440

  9. Epithelial TRPV1 signaling accelerates gingival epithelial cell proliferation.

    PubMed

    Takahashi, N; Matsuda, Y; Yamada, H; Tabeta, K; Nakajima, T; Murakami, S; Yamazaki, K

    2014-11-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca(2+) levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  10. Magnetite induces oxidative stress and apoptosis in lung epithelial cells.

    PubMed

    Ramesh, Vani; Ravichandran, Prabakaran; Copeland, Clinton L; Gopikrishnan, Ramya; Biradar, Santhoshkumar; Goornavar, Virupaxi; Ramesh, Govindarajan T; Hall, Joseph C

    2012-04-01

    There is an ongoing concern regarding the biocompatibility of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. In this study, we investigated the toxic properties of magnetite stabilized with polyacrylate sodium. The magnetite was characterized by X-ray powder diffraction analysis, and the mean particle diameter was calculated using the Scherrer formula and was found to be 9.3 nm. In this study, we treated lung epithelial cells with different concentrations of magnetite and investigated their effects on oxidative stress and cell proliferation. Our data showed an inhibition of cell proliferation in magnetite-treated cells with a significant dose-dependent activation and induction of reactive oxygen species. Also, we observed a depletion of antioxidants, glutathione, and superoxide dismutase, respectively, as compared with control cells. In addition, apoptotic-related protease/enzyme such as caspase-3 and -8 activities, were increased in a dose-dependent manner with corresponding increased levels of DNA fragmentation in magnetite-treated cells compared to than control cells. Together, the present study reveals that magnetite exposure induces oxidative stress and depletes antioxidant levels in the cells to stimulate apoptotic pathway for cell death. PMID:22147200

  11. Anti-Apoptotic Gene Delivery with cyclo-(d-Trp-Tyr) Peptide Nanotube via Eye Drop Following Corneal Epithelial Debridement

    PubMed Central

    Lee, Yu-Hsing; Chang, Shwu-Fen; Liaw, Jiahorng

    2015-01-01

    Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carriers on corneal injury. First, the PNTs were found within 2 μm in length and 300 nm in width by an atomic force microscope and confocal laser microscope system. Plasmid DNAs were observed to be associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were associated with tyrosine of PNTs with a binding constant of 2.7 × 108 M−1. The stability of plasmid DNA with PNTs against the DNase was found at 60 min. Using thioflavin T pre-stained PNTs on the corneal eye drop delivery, the distribution of PNTs was in the epithelial and stroma regions. After corneal debridement, the rhodamine-labeled plasmid DNA and thioflavin T pre-stained PNTs were also delivered and could be observed in the stroma of cornea. PNTs complexed with anti-apoptotic plasmid caspase 3 silencing shRNA eye drop delivery decreased 41% of caspase 3 activity after the first dose by caspase 3 activity and Western blot analysis. PMID:26193308

  12. Differentiation capacity of epithelial cells in the sponge Suberites domuncula.

    PubMed

    Schröder, Heinz C; Perović-Ottstadt, Sanja; Wiens, Matthias; Batel, Renato; Müller, Isabel M; Müller, Werner E G

    2004-05-01

    Sponges (phylum Porifera) represent the oldest metazoans. Their characteristic metazoan adhesion molecules and transcription factors enable them to establish a complex "Bauplan"; three major differentiated cell types (epithelial cells, skeletal cells/sclerocytes, and contractile cells) can be distinguished. Since no molecular markers are as yet available to distinguish these somatic cells or the corresponding embryonic cells from which they originate, we have selected the following three genes for their characterization: noggin (a signaling molecule in development), a caspase that encodes an apoptotic molecule, and silicatein. Silicatein is an enzyme that is involved in the synthesis of siliceous spicules and can hence be considered as a marker for scleroblasts. We have used the demosponge Suberites domuncula as a model system. During the hatching of the gemmules (asexual reproduction bodies) of S. domuncula, the expression of both noggin and caspase increases, whereas no transcripts for silicatein can be detected, irrespective of the presence of silicate or ferric iron (Fe3+) in the medium. In contrast, in adult specimens, silicate/Fe3+ cause an increased expression of these genes. In situ analysis has revealed that the first cells that express noggin, caspase, and silicatein lie in the epithelial layer of the pinacoderm. In a later phase, the noggin- and silicatein-positive cells migrate into the mesohyl, where they are found in association with spicules. Thus, the pinacoderm of sponges contains cells that have a differentiating capacity and from which somatic cells, such as skeletal cells/sclerocytes, derive. PMID:15024642

  13. Interaction of Late Apoptotic and Necrotic Cells with Vitronectin

    PubMed Central

    Stepanek, Ondrej; Brdicka, Tomas; Angelisova, Pavla; Horvath, Ondrej; Spicka, Jiri; Stockbauer, Petr; Man, Petr; Horejsi, Vaclav

    2011-01-01

    Background Vitronectin is an abundant plasma glycoprotein identified also as a part of extracellular matrix. Vitronectin is substantially enriched at sites of injured, fibrosing, inflamed, and tumor tissues where it is believed to be involved in wound healing and tissue remodeling. Little is known about the mechanism of vitronectin localization into the damaged tissues. Methodology/Principal Findings 2E12 antibody has been described to bind a subset of late apoptotic cells. Using immunoisolation followed by mass spectrometry, we identified the antigen recognized by 2E12 antibody as vitronectin. Based on flow cytometry, we described that vitronectin binds to the late apoptotic and necrotic cells in cell cultures in vitro as well as in murine thymus and spleen in vivo. Confocal microscopy revealed that vitronectin binds to an intracellular cytoplasmic structure after the membrane rupture. Conclusions/Significance We propose that vitronectin could serve as a marker of membrane disruption in necrosis and apoptosis for flow cytometry analysis. Moreover, we suggest that vitronectin binding to dead cells may represent one of the mechanisms of vitronectin incorporation into the injured tissues. PMID:21573223

  14. Symmetry breaking mechanism for epithelial cell polarization

    NASA Astrophysics Data System (ADS)

    Veglio, A.; Gamba, A.; Nicodemi, M.; Bussolino, F.; Serini, G.

    2009-09-01

    In multicellular organisms, epithelial cells form layers separating compartments responsible for different physiological functions. At the early stage of epithelial layer formation, each cell of an aggregate defines an inner and an outer side by breaking the symmetry of its initial state, in a process known as epithelial polarization. By integrating recent biochemical and biophysical data with stochastic simulations of the relevant reaction-diffusion system, we provide evidence that epithelial cell polarization is a chemical phase-separation process induced by a local bistability in the signaling network at the level of the cell membrane. The early symmetry breaking event triggering phase separation is induced by adhesion-dependent mechanical forces localized in the point of convergence of cell surfaces when a threshold number of confluent cells is reached. The generality of the emerging phase-separation scenario is likely common to many processes of cell polarity formation.

  15. Airway epithelial cell responses to ozone injury

    SciTech Connect

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

    1995-03-01

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

  16. In vitro apoptotic cell death during erythroid differentiation.

    PubMed

    Zamai, L; Burattini, S; Luchetti, F; Canonico, B; Ferri, P; Melloni, E; Gonelli, A; Guidotti, L; Papa, S; Falcieri, E

    2004-03-01

    Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro. PMID:15004520

  17. Effects of butyrate on the expression of insulin-like growth factor binding proteins in bovine kidney epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sodium butyrate induces cell cycle arrest and apoptosis in bovine kidney epithelial cells primarily via down-regulating cell cycle-related gene expression and enhancing expression of pro-apoptotic genes. The insulin-like growth factor (IGF) system plays an essential role in these processes as well a...

  18. Apoptotic cells are cleared by directional migration and elmo1- dependent macrophage engulfment.

    PubMed

    van Ham, Tjakko J; Kokel, David; Peterson, Randall T

    2012-05-01

    Apoptotic cell death is essential for development and tissue homeostasis. Failure to clear apoptotic cells can ultimately cause inflammation and autoimmunity. Apoptosis has primarily been studied by staining of fixed tissue sections, and a clear understanding of the behavior of apoptotic cells in living tissue has been elusive. Here, we use a newly developed technique to track apoptotic cells in real time as they emerge and are cleared from the zebrafish brain. We find that apoptotic cells are remarkably motile, frequently migrating several cell diameters to the periphery of living tissues. F-actin remodeling occurs in surrounding cells, but also within the apoptotic cells themselves, suggesting a cell-autonomous component of motility. During the first 2 days of development, engulfment is rare, and most apoptotic cells lyse at the brain periphery. By 3 days postfertilization, most cell corpses are rapidly engulfed by macrophages. This engulfment requires the guanine nucleotide exchange factor elmo1. In elmo1-deficient macrophages, engulfment is rare and may occur through macropinocytosis rather than directed engulfment. These findings suggest that clearance of apoptotic cells in living vertebrates is accomplished by the combined actions of apoptotic cell migration and elmo1-dependent macrophage engulfment. PMID:22503503

  19. Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

    PubMed Central

    Ponomarev, Eugeny D.; Tsytsykova, Alla; Armant, Myriam; Vorobjev, Ivan A.

    2014-01-01

    High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE+ cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations. PMID:24394470

  20. Reducing VDAC1 expression induces a non-apoptotic role for pro-apoptotic proteins in cancer cell differentiation.

    PubMed

    Arif, Tasleem; Krelin, Yakov; Shoshan-Barmatz, Varda

    2016-08-01

    Proteins initially identified as essential for apoptosis also mediate a wide range of non-apoptotic functions that include cell cycle progression, differentiation and metabolism. As this phenomenon was mostly reported with non-cancer cells, we considered non-conventional roles for the apoptotic machinery in the cancer setting. We found that treating glioblastoma (GBM) tumors with siRNA against VDAC1, a mitochondrial protein found at the crossroads of metabolic and survival pathways and involved in apoptosis, inhibited tumor growth while leading to differentiation of tumor cells into neuronal-like cells, as reflected in the expression of specific markers. Although VDAC1 depletion did not induce apoptosis, the expression levels of several pro-apoptotic regulatory proteins were changed. Specifically, VDAC1 deletion led to up-regulation of caspases, p53, cytochrome c, and down-regulation of SMAC/Diablo, AIF and TSPO. The down-regulated group was highly expressed in U-87MG xenografts, as well as in GBMs from human patients. We also showed that the rewired cancer-cell metabolism resulting from VDAC1 depletion reinforced cell growth arrest and differentiation via alterations in the transcription factors p53, c-Myc, HIF-1α and NF-κB. The decrease in c-Myc, HIF-1α and NF-κB levels was in accord with reduced cell proliferation, whereas increased p53 expression promoted differentiation. Thus, upon metabolic re-programing induced by VDAC1 depletion, the levels of pro-apoptotic proteins associated with cell growth decreased, while those connected to cell differentiation increased, converting GBM cells into astrocyte- and neuron-like cells. The results reveal that in tumors, pro-apoptotic proteins can perform non-apoptotic functions, acting as regulators of cell growth and differentiation, making these molecules potential new targets for cancer therapy. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy

  1. The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity

    PubMed Central

    Ayala, Inmaculada; Colanzi, Antonino; Lapazio, Lucia; Corda, Daniela; Soriani, Marco; Pizza, Mariagrazia; Rossi Paccani, Silvia

    2015-01-01

    Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis. PMID:25996923

  2. Epithelial Cell Proliferation Contributes to Airway Remodeling in Severe Asthma

    PubMed Central

    Cohen, Lance; E, Xueping; Tarsi, Jaime; Ramkumar, Thiruvamoor; Horiuchi, Todd K.; Cochran, Rebecca; DeMartino, Steve; Schechtman, Kenneth B.; Hussain, Iftikhar; Holtzman, Michael J.; Castro, Mario

    2007-01-01

    Rationale: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction. Objectives: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling. Methods: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis. Measurements and Main Results: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p = 0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p = 0.002) and Ki67 was increased (p < 0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p < 0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p = 0.002), suggesting increased cell death. Conclusions: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe

  3. Apoptotic effect of noscapine in breast cancer cell lines.

    PubMed

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  4. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-01

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes. PMID:26859350

  5. Persistence of apoptotic cells without autoimmune disease or inflammation in CD14−/− mice

    PubMed Central

    Devitt, Andrew; Parker, Kate G.; Ogden, Carol Anne; Oldreive, Ceri; Clay, Michael F.; Melville, Lynsey A.; Bellamy, Christopher O.; Lacy-Hulbert, Adam; Gangloff, Sophie C.; Goyert, Sanna M.; Gregory, Christopher D.

    2004-01-01

    Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14−/− macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14−/− macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences. PMID:15611337

  6. ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis

    PubMed Central

    Guo, Feiye; Ding, Ying; Caberoy, Nora; Alvarado, Gabriela; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis of shed photoreceptor outer segments (POSs) by retinal pigment epithelial (RPE) cells is critical to retinal homeostasis and shares many conserved signaling pathways with other phagocytes, including extrinsic regulations. Phagocytotic ligands are the key to cargo recognition, engulfment initiation, and activity regulation. In this study, we identified intracellular protein ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytotic ligand by a new approach of functional screening. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cells. This finding was further corroborated with primary RPE cells and RPE explants. Internalized POS vesicles were colocalized with a phagosome marker, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells and selectively bound to shed POS vesicles and apoptotic cells, possibly via externalized phosphatidylserine. ABCF1 is predominantly expressed in POSs and colocalized with the POS marker rhodopsin, providing geographical convenience for regulation of RPE phagocytosis. Collectively these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway. Furthermore, the new approach is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to understand extrinsic regulation and cargo recognition. PMID:25904329

  7. Non-apoptotic cell death associated with perturbations of macropinocytosis

    PubMed Central

    Maltese, William A.; Overmeyer, Jean H.

    2015-01-01

    Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed “methuosis,” from the Greek methuo (to drink to intoxication). It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms. PMID:25762935

  8. Odontogenic epithelial stem cells: hidden sources.

    PubMed

    Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

    2015-12-01

    The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed. PMID:26367485

  9. Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

    NASA Astrophysics Data System (ADS)

    Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

    2013-12-01

    Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

  10. Novel human bronchial epithelial cell lines for cystic fibrosis research

    PubMed Central

    Fulcher, M. L.; Gabriel, S. E.; Olsen, J. C.; Tatreau, J. R.; Gentzsch, M.; Livanos, E.; Saavedra, M. T.; Salmon, P.; Randell, S. H.

    2009-01-01

    Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ΔF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Ω·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1β, TNF-α, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development. PMID:18978040

  11. Signal transduction induced by apoptotic cells inhibits HIV transcription in monocytes/macrophages.

    PubMed

    Gekonge, Bethsebah N; Schiralli, Gillian; Schlegel, Robert A; Henderson, Andrew J

    2006-10-01

    The primary targets of HIV are CD4(+) T cells and macrophages. HIV infection is associated with an increase in apoptosis of infected and uninfected CD4(+) T cells, and these infected cells undergo apoptosis and produce HIV virions with phosphatidylserine (PS) on their surface. During phagocytosis of apoptotic cells, macrophages, using an array of receptors, are able to perceive various surface changes on apoptotic cells. The engagement of phagocytic receptors by ligands on the apoptotic cell surface results in the activation of signaling cascades, which facilitate engulfment. In this study, we examined how PS associated with virions and apoptotic cells influences HIV replication. We demonstrate that virus-associated PS is required for HIV infection of macrophages at a step prior to integration but following strong-stop, indicating that PS-initiated signals alter the establishment of HIV provirus. Conversely, apoptotic cells inhibited HIV transcription in infected macrophages, although this ability to suppress transcription was independent of PS. Furthermore, we show that ELMO, a key signaling molecule that participates in the phagocytosis of apoptotic cells, inhibited HIV transcription; however, knocking down endogenous ELMO expression in infected U937 cells rescued HIV transcription when these cells were coincubated with apoptotic targets. Taken together, these data show that apoptotic cells and the signals, which they initiate upon recognition by macrophages, influence the successful establishment of HIV infection and provirus transcription. PMID:16885500

  12. Two roads to death - Bax targets mitochondria by distinct routes before or during apoptotic cell death.

    PubMed

    Dewson, Grant

    2015-01-01

    Recent studies have revolutionized our understanding of how the crucial apoptosis effectors Bax and Bak target mitochondria to kill cells. We recently reported that an important determinant of the localization, oligomerization, and apoptotic function of Bax is an interaction with either mitochondrial voltage-dependent anion channel 2 (VDAC2) (in healthy cells) or Bak (in apoptotic cells).(1). PMID:27308408

  13. Apoptotic cell-based therapies against transplant rejection: role of recipient’s dendritic cells

    PubMed Central

    Larregina, Adriana T.

    2010-01-01

    One of the ultimate goals in transplantation is to develop novel therapeutic methods for induction of donor-specific tolerance to reduce the side effects caused by the generalized immunosuppression associated to the currently used pharmacologic regimens. Interaction or phagocytosis of cells in early apoptosis exerts potent anti-inflammatory and immunosuppressive effects on antigen (Ag)-presenting cells (APC) like dendritic cells (DC) and macrophages. This observation led to the idea that apoptotic cell-based therapies could be employed to deliver donor-Ag in combination with regulatory signals to recipient’s APC as therapeutic approach to restrain the anti-donor response. This review describes the multiple mechanisms by which apoptotic cells down-modulate the immuno-stimulatory and pro-inflammatory functions of DC and macrophages, and the role of the interaction between apoptotic cells and APC in self-tolerance and in apoptotic cell-based therapies to prevent/treat allograft rejection and graft-versus-host disease in murine experimental systems and in humans. It also explores the role that in vivo-generated apoptotic cells could have in the beneficial effects of extracorporeal photopheresis, donor-specific transfusion, and tolerogenic DC-based therapies in transplantation. PMID:20140521

  14. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells.

    PubMed

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  15. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    PubMed Central

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  16. Concise Review: Apoptotic Cell-Based Therapies-Rationale, Preclinical Results and Future Clinical Developments.

    PubMed

    Saas, Philippe; Daguindau, Etienne; Perruche, Sylvain

    2016-06-01

    The objectives of this review are to summarize the experimental data obtained using apoptotic cell-based therapies, and then to discuss future clinical developments. Indeed, apoptotic cells exhibit immunomodulatory properties that are reviewed here by focusing on more recent mechanisms. These immunomodulatory mechanisms are in particular linked to the clearance of apoptotic cells (called also efferocytosis) by phagocytes, such as macrophages, and the induction of regulatory T cells. Thus, apoptotic cell-based therapies have been used to prevent or treat experimental inflammatory diseases. Based on these studies, we have identified critical steps to design future clinical trials. This includes: the administration route, the number and schedule of administration, the appropriate apoptotic cell type to be used, as well as the apoptotic signal. We also have analyzed the clinical relevancy of apoptotic-cell-based therapies in experimental models. Additional experimental data are required concerning the treatment of inflammatory diseases (excepted for sepsis) before considering future clinical trials. In contrast, apoptotic cells have been shown to favor engraftment and to reduce acute graft-versus-host disease (GvHD) in different relevant models of transplantation. This has led to the conduct of a phase 1/2a clinical trial to alleviate GvHD. The absence of toxic effects obtained in this trial may support the development of other clinical studies based on this new cell therapy. Stem Cells 2016;34:1464-1473. PMID:27018198

  17. Induced pluripotency of human prostatic epithelial cells.

    PubMed

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

    2013-01-01

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

  18. Single cell analysis demonstrates how nutrient deprivation creates apoptotic and quiescent cell populations in tumor cylindroids

    PubMed Central

    Kim, Byoung-jin; Forbes, Neil S.

    2016-01-01

    Understanding how quiescent and apoptotic populations form in tumors is necessary because these cell types can considerably diminish therapeutic efficacy. Most cancer therapeutics are ineffective against quiescent cells because they target rapidly proliferating cells. Distinguishing apoptosis is important because apoptotic cells are committed to death and do not require treatment. Regrowth of quiescent cell can lead to tumor reoccurrence and metastasis, which are the leading causes of cancer mortality. We hypothesized that cylindroid cultures and acridine orange staining could be used to determine how nutrient diffusion creates apoptotic and quiescent regions in tumors. To test this hypothesis we developed a microscopy technique to measure cellular DNA and RNA content in single cells using thin cylindroids and acridine orange staining. Cell classification was compared to flow cytometry of cells grown in defined monolayer cultures. The presence of apoptosis was confirmed by morphological nuclear analysis. The effect of diffusion was determined by varying incubation time, cylindroid size, and exposing cylindroids to nutrient-deficient media. Four overlapping regions were identified as a function of cylindroid radius: an outer viable/quiescent region; a second quiescent/apoptotic region; a third late-stage apoptotic region; and an inner dead region. In monolayer cultures the absence of glutamine and growth factors induced apoptosis and hypoxia induced quiescence. Treating with nutrient-deficient media suggested that cells became quiescent near the periphery because of glucose and oxygen limitations, and became apoptotic and died further from the edge because of glutamine and growth factor limitations. These results show that cellular microenvironments can be identified in cylindroids using simple acridine orange staining and that single cell florescence can be measured in three-dimensional culture. The developed techniques will be useful for developing cancer

  19. Sustained Polymorphonuclear Leukocyte Transmigration Induces Apoptosis in T84 Intestinal Epithelial Cells

    PubMed Central

    Le'Negrate, Gaëlle; Selva, Eric; Auberger, Patrick; Rossi, Bernard; Hofman, Paul

    2000-01-01

    Acute colitis is characterized by a large number of polymorphonuclear leukocytes (PMNLs) migrating across the columnar epithelium in response to inflammatory stimuli. Several of these inflammatory factors have been characterized as proapoptotic inducers for intestinal epithelial cells. Our aim was to elucidate the role of PMNL transmigration in the onset of intestinal epithelial cell apoptosis. We found that PMNL migration, in response to N-formyl-methionyl-leucyl-phenylalanine across monolayers of intestinal epithelial cells (T84), was associated with activation of caspase-2, -3, and -9 and poly(ADP-ribose) polymerase cleavage within epithelial cells. Moreover, dihydrocytochalasin B treatment of T84 cells induced apoptosis with similar characteristics. Although Fas and Fas ligand were expressed on T84 cells and PMNLs, treatment of epithelial cells with an antagonistic anti-Fas antibody failed to prevent apoptosis induced by migrating PMNLs. Owing to the F-actin reorganization accompanying PMNL transmigration, these findings indicate a direct relationship between PMNL migration and induction of apoptosis in epithelial cells. This apoptotic process appears to involve remodeling of the actin cytoskeleton of enterocytes independent of the Fas/Fas ligand pathway. PMID:10995451

  20. Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells

    PubMed Central

    Chen, Congcong; Dienhart, Jason A.; Bolton, Eric C.

    2016-01-01

    Androgen receptor (AR) signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. 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 have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT) treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS), TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP) in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2) were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1) were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins, such that

  1. Epithelial Cell Shedding and Barrier Function

    PubMed Central

    Williams, J. M.; Duckworth, C. A.; Burkitt, M. D.; Watson, A. J. M.; Campbell, B. J.

    2015-01-01

    The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed. PMID:25428410

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

    EPA Science Inventory

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

  3. Epithelial Cell Regulation of Allergic Diseases.

    PubMed

    Gour, Naina; Lajoie, Stephane

    2016-09-01

    Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental stimuli. Why only certain individuals mount inappropriate type 2 immune responses to these otherwise harmless allergens has remained an unanswered question. Mounting evidence suggests that the epithelium, by sensing its environment, is the central regulator of allergic diseases. Once considered to be a passive barrier to allergens, epithelial cells at mucosal surfaces are now considered to be the cornerstone of the allergic diathesis. Beyond their function as maintaining barrier at mucosal surfaces, mucosal epithelial cells through the secretion of mediators like IL-25, IL-33, and TSLP control the fate of downstream allergic immune responses. In this review, we will discuss the advances in recent years regarding the process of allergen recognition and secretion of soluble mediators by epithelial cells that shape the development of the allergic response. PMID:27534656

  4. Apoptotic cells induce dendritic cell-mediated suppression via interferon-γ-induced IDO

    PubMed Central

    Williams, Charlotte A; Harry, Rachel A; McLeod, Julie D

    2008-01-01

    Dendritic cells (DC) are sensitive to their local environment and are affected by proximal cell death. This study investigated the modulatory effect of cell death on DC function. Monocyte-derived DC exposed to apoptotic Jurkat or primary T cells failed to induce phenotypic maturation of the DC and were unable to support CD4+ allogeneic T-cell proliferation compared with DC exposed to lipopolysaccharide (LPS) or necrotic cells. Apoptotic cells coincubated with LPS- or necrotic cell-induced mature DC significantly suppressed CD80, CD86 and CD83 and attenuated LPS-induced CD4+ T-cell proliferation. Reduced levels of interleukin-12 (IL-12), IL-10, IL-6, tumour necrosis factor-α and interferon-γ (IFN-γ) were found to be concomitant with the suppressive activity of apoptotic cells upon DC. Furthermore, intracellular staining confirmed IFN-γ expression by DC in association with apoptotic environments. The specific generation of IFN-γ by DC within apoptotic environments is suggestive of an anti-inflammatory role by the induction of indoleamine 2,3-dioxygenase (IDO). Both neutralization of IFN-γ and IDO blockade demonstrated a role for IFN-γ and IDO in the suppression of CD4+ T cells. Moreover, we demonstrate that IDO expression within the DC was found to be IFN-γ-dependent. Blocking transforming growth factor-β (TGF-β) also produced a partial release in T-cell proliferation. Our study strongly suggests that apoptosis-induced DC suppression is not an immunological null event and two prime mediators underpinning these functional effects are IFN-γ-induced IDO and TGF-β. PMID:18067553

  5. Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

    PubMed

    Goto, Yoshiyuki; Obata, Takashi; Kunisawa, Jun; Sato, Shintaro; Ivanov, Ivaylo I; Lamichhane, Aayam; Takeyama, Natsumi; Kamioka, Mariko; Sakamoto, Mitsuo; Matsuki, Takahiro; Setoyama, Hiromi; Imaoka, Akemi; Uematsu, Satoshi; Akira, Shizuo; Domino, Steven E; Kulig, Paulina; Becher, Burkhard; Renauld, Jean-Christophe; Sasakawa, Chihiro; Umesaki, Yoshinori; Benno, Yoshimi; Kiyono, Hiroshi

    2014-09-12

    Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation. PMID:25214634

  6. Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1.

    PubMed

    Fond, Aaron M; Lee, Chang Sup; Schulman, Ira G; Kiss, Robert S; Ravichandran, Kodi S

    2015-07-01

    Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane-initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell-initiated pathway functioned independently of the liver X receptor (LXR) sterol-sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo. PMID:26075824

  7. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    SciTech Connect

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

  8. Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1

    PubMed Central

    Fond, Aaron M.; Lee, Chang Sup; Schulman, Ira G.; Kiss, Robert S.; Ravichandran, Kodi S.

    2015-01-01

    Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane–initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell–initiated pathway functioned independently of the liver X receptor (LXR) sterol–sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo. PMID:26075824

  9. UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

    PubMed

    Juncker, Tom; Cerella, Claudia; Teiten, Marie-Hélène; Morceau, Franck; Schumacher, Marc; Ghelfi, Jenny; Gaascht, François; Schnekenburger, Michael; Henry, Estelle; Dicato, Mario; Diederich, Marc

    2011-01-01

    Cardiac steroids are used to treat various diseases including congestive heart failure and cancer. The aim of this study was to investigate the anti-leukemic activity of UNBS1450, a hemi-synthetic cardenolide belonging to the cardiac steroid glycoside family. Here, we report that, at low nanomolar concentrations, UNBS1450 induces apoptotic cell death. Subsequently, we have investigated the molecular mechanisms leading to apoptosis activation. Our results show that UNBS1450 inhibits NF-κB transactivation and triggers apoptosis by cleavage of pro-caspases 8, 9 and 3/7, by decreasing expression of anti-apoptotic Mcl-1 and by recruitment of pro-apoptotic Bak and Bax protein eventually resulting in cell death. PMID:20849830

  10. Phagocytosis mechanism of apoptotic granulosa cells regulated by milk-fat globule-EGF factor 8.

    PubMed

    Naka, Mayumi; Kusakabe, Ken; Takeshita, Ai; Nakagawa, Hiroshi; Ito, Yuko; Shibata, Masa-Aki; Otsuki, Yoshinori

    2009-09-01

    In the process of ovary sexual maturation, most immature ovarian follicles degrade into atretic follicles accompanied by apoptosis in granulosa cells. Macrophages can recognize apoptotic cells through specific binding with phosphatidylserine (PS), exposed on the surface of apoptotic cells, which is mediated by milk-fat globule-EGF factor 8 (MFG-E8). In the present research, we examined the involvement of the MFG-E8-dependent phagocytosis system in the atretic follicles of developing mouse ovaries. The number of atretic follicles and DNA-fragmented granulosa cells significantly increased in B6C3F1 mice during 2 to 6 weeks. Chromatin-condensed granulosa cells were engulfed by macrophages, which existed in the stroma or atretic follicles, or by neighboring normal granulosa cells. MFG-E8 mRNA increased in ovaries during 2 to 6 weeks, and immunoreactivity of MFG-E8 was detected at the surface of apoptotic cells existing around the antrum. Immunoelectron microscopic study revealed MFG-E8-positive signals on the membrane of apoptotic cells near macrophages, but apoptotic cells engulfed by neighboring granulosa cells showed few signals. Anti-Fas antibody elevated the annexin-V-positive reaction in isolated granulosa cells from 3-week-old mouse ovaries. MFG-E8 seems to act on the phagocytosis of apoptotic granulosa cells via macrophages and contribute to the regression process of atretic follicles. PMID:19784740

  11. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

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

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  12. Sulbutiamine counteracts trophic factor deprivation induced apoptotic cell death in transformed retinal ganglion cells.

    PubMed

    Kang, Kui Dong; Majid, Aman Shah Abdul; Kim, Kyung-A; Kang, Kyungsu; Ahn, Hong Ryul; Nho, Chu Won; Jung, Sang Hoon

    2010-11-01

    Sulbutiamine is a highly lipid soluble synthetic analogue of vitamin B(1) and is used clinically for the treatment of asthenia. The aim of our study was to demonstrate whether sulbutiamine is able to attenuate trophic factor deprivation induced cell death to transformed retinal ganglion cells (RGC-5). Cells were subjected to serum deprivation for defined periods and sulbutiamine at different concentrations was added to the cultures. Various procedures (e.g. cell viability assays, apoptosis assay, reactive oxygen species analysis, Western blot analysis, flow cytometric analysis, glutathione (GSH) and glutathione-S-transferase (GST) measurement) were used to demonstrate the effect of sulbutiamine. Sulbutiamine dose-dependently attenuated apoptotic cell death induced by serum deprivation and stimulated GSH and GST activity. Moreover, sulbutiamine decreased the expression of cleaved caspase-3 and AIF. This study demonstrates for the first time that sulbutiamine is able to attenuate trophic factor deprivation induced apoptotic cell death in neuronal cells in culture. PMID:20809085

  13. Respiratory epithelial cells orchestrate pulmonary innate immunity.

    PubMed

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

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

  14. Bioactive compounds from crocodile (Crocodylus siamensis) white blood cells induced apoptotic cell death in hela cells.

    PubMed

    Patathananone, Supawadee; Thammasirirak, Sompong; Daduang, Jureerut; Chung, Jing Gung; Temsiripong, Yosapong; Daduang, Sakda

    2016-08-01

    Crocodile (Crocodylus siamensis) white blood cell extracts (WBCex) were examined for anticancer activity in HeLa cell lines using the MTT assay. The percentage viability of HeLa cells significantly deceased after treatment with WBCex in a dose- and time-dependent manner. The IC50 dose was suggested to be approximately 225 μg/mL protein. Apoptotic cell death occurred in a time-dependent manner based on investigation by flow cytometry using annexin V-FITC and PI staining. DAPI nucleic acid staining indicated increased chromatin condensation. Caspase-3, -8 and -9 activities also increased, suggesting the induction of the caspase-dependent apoptotic pathway. Furthermore, the mitochondrial membrane potential (ΔΨm ) of HeLa cells was lost as a result of increasing levels of Bax and reduced levels of Bcl-2, Bcl-XL, Bcl-Xs, and XIAP. The decreased ΔΨm led to the release of cytochrome c and the activation of caspase-9 and -3. Apoptosis-inducing factor translocated into the nuclei, and endonuclease G (Endo G) was released from the mitochondria. These results suggest that anticancer agents in WBCex can induce apoptosis in HeLa cells via both caspase-dependent and -independent pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 986-997, 2016. PMID:25691005

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

    PubMed

    O'Connell, J; Bennett, M W; Nally, K; O'Sullivan, G C; Collins, J K; Shanahan, F

    2000-12-01

    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. PMID:11056003

  16. Gene delivery of the elastase inhibitor elafin protects macrophages from neutrophil elastase-mediated impairment of apoptotic cell recognition.

    PubMed

    Henriksen, Peter A; Devitt, Andrew; Kotelevtsev, Yuri; Sallenave, Jean-Michel

    2004-09-10

    The resolution of inflammation is dependent on recognition and phagocytic removal of apoptotic cells by macrophages. Receptors for apoptotic cells are sensitive to degradation by human neutrophil elastase (HNE). We show in the present study that HNE cleaves macrophage cell surface CD14 and in so doing, reduces phagocytic recognition of apoptotic lymphocytic cells (Mutu 1). Using an improved method of adenovirus-mediated transfection of macrophages with the HNE inhibitor elafin, we demonstrate that elafin overexpression prevents CD14 cleavage and restores apoptotic cell recognition by macrophages. This approach of genetic modification of macrophages could be used to restore apoptotic cell recognition in inflammatory conditions. PMID:15358543

  17. Gambogic acid induces apoptotic cell death in T98G glioma cells.

    PubMed

    Thida, Mya; Kim, Dae Won; Tran, Thi Thu Thuy; Pham, Minh Quan; Lee, Heesu; Kim, Inki; Lee, Jae Wook

    2016-02-01

    Gambogic acid (GA), a natural product with a xanthone structure, has a broad range of anti-proliferative effects on cancer cell lines. We evaluated GA for its cytotoxic effects on T98G glioblastoma cells. GA exhibited potent anti-proliferative activity and induced apoptosis in T98G glioblastoma cells in a dose-dependent manner. Incubation of cells with GA revealed apoptotic features including increased Bax and AIF expression, cytochrome c release, and cleavage of caspase-3, -8, -9, and PARP, while Bcl-2 expression was downregulated. Furthermore, GA induced reactive oxygen species (ROS) generation in T98G cells. Our results indicate that GA increases Bax- and AIF-associated apoptotic signaling in glioblastoma cells. PMID:26631318

  18. The proteasomal and apoptotic phenotype determine bortezomib sensitivity of non-small cell lung cancer cells

    PubMed Central

    Voortman, Jens; Chęcińska, Agnieszka; Giaccone, Giuseppe

    2007-01-01

    Bortezomib is a novel anti-cancer agent which has shown promising activity in non-small lung cancer (NSCLC) patients. However, only a subset of patients respond to this treatment. We show that NSCLC cell lines are differentially sensitive to bortezomib, IC50 values ranging from 5 to 83 nM. The apoptosis-inducing potential of bortezomib in NSCLC cells was found to be dependent not only on the apoptotic phenotype but also on the proteasomal phenotype of individual cell lines. Upon effective proteasome inhibition, H460 cells were more susceptible to apoptosis induction by bortezomib than SW1573 cells, indicating a different apoptotic phenotype. However, exposure to a low dose of bortezomib did only result in SW1573 cells, and not in H460 cells, in inhibition of proteasome activity and subsequent apoptosis. This suggests a different proteasomal phenotype as well. Additionally, overexpression of anti-apoptotic protein Bcl-2 in H460 cells did not affect the proteasomal phenotype of H460 cells but did result in decreased bortezomib-induced apoptosis. In conclusion, successful proteasome-inhibitor based treatment strategies in NSCLC face the challenge of having to overcome apoptosis resistance as well as proteasomal resistance of individual lung cancer cells. Further studies in NSCLC are warranted to elucidate underlying mechanisms. PMID:18021420

  19. Serum-derived plasminogen is activated by apoptotic cells and promotes their phagocytic clearance.

    PubMed

    Rosenwald, Matthias; Koppe, Uwe; Keppeler, Hildegard; Sauer, Guido; Hennel, Roman; Ernst, Anne; Blume, Karin Erika; Peter, Christoph; Herrmann, Martin; Belka, Claus; Schulze-Osthoff, Klaus; Wesselborg, Sebastian; Lauber, Kirsten

    2012-12-15

    The elimination of apoptotic cells, called efferocytosis, is fundamentally important for tissue homeostasis and prevents the onset of inflammation and autoimmunity. Serum proteins are known to assist in this complex process. In the current study, we performed a multistep chromatographic fractionation of human serum and identified plasminogen, a protein involved in fibrinolysis, wound healing, and tissue remodeling, as a novel serum-derived factor promoting apoptotic cell removal. Even at levels significantly lower than its serum concentration, purified plasminogen strongly enhanced apoptotic prey cell internalization by macrophages. Plasminogen acted mainly on prey cells, whereas on macrophages no enhancement of the engulfment process was observed. We further demonstrate that the efferocytosis-promoting activity essentially required the proteolytic activation of plasminogen and was completely abrogated by the urokinase plasminogen activator inhibitor-1 and serine protease inhibitor aprotinin. Thus, our study assigns a new function to plasminogen and plasmin in apoptotic cell clearance. PMID:23150713

  20. Phototherapy-treated apoptotic tumor cells induce pro-inflammatory cytokines production in macrophage

    NASA Astrophysics Data System (ADS)

    Lu, Cuixia; Wei, Yanchun; Xing, Da

    2014-09-01

    Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high fluence low-power laser irradiation (HF-LPLI) induces mitochondrial superoxide anion burst, resulting in oxidative damage to tumor cells. In this study, we further explored the immunological effects of HF-LPLI-induced apoptotic tumor cells. When macrophages were co-incubated with apoptotic cells induced by HF-LPLI, we observed the increased levels of TNF-α secretion and NO production in macrophages. Further experiments showed that NF-κB was activated in macrophages after co-incubation with HF-LPLI-induced apoptotic cells, and inhibition of NF-κB activity by pyrrolidinedithiocarbamic acid (PDTC) reduced the elevated levels of TNF-α secretion and NO production. These data indicate that HF-LPLI-induced apoptotic tumor cells induce the secretion of pro-inflammatory cytokines in macrophages, which may be helpful for better understanding the biological effects of cancer phototherapy.

  1. Die another way – non-apoptotic mechanisms of cell death

    PubMed Central

    Tait, Stephen W. G.; Ichim, Gabriel; Green, Douglas R.

    2014-01-01

    ABSTRACT Regulated, programmed cell death is crucial for all multicellular organisms. Cell death is essential in many processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells. The best-studied form of programmed cell death is apoptosis, a process that requires activation of caspase proteases. Recently it has been appreciated that various non-apoptotic forms of cell death also exist, such as necroptosis and pyroptosis. These non-apoptotic cell death modalities can be either triggered independently of apoptosis or are engaged should apoptosis fail to execute. In this Commentary, we discuss several regulated non-apoptotic forms of cell death including necroptosis, autophagic cell death, pyroptosis and caspase-independent cell death. We outline what we know about their mechanism, potential roles in vivo and define outstanding questions. Finally, we review data arguing that the means by which a cell dies actually matters, focusing our discussion on inflammatory aspects of cell death. PMID:24833670

  2. Oncogenic Properties of Apoptotic Tumor Cells in Aggressive B Cell Lymphoma

    PubMed Central

    Ford, Catriona A.; Petrova, Sofia; Pound, John D.; Voss, Jorine J.L.P.; Melville, Lynsey; Paterson, Margaret; Farnworth, Sarah L.; Gallimore, Awen M.; Cuff, Simone; Wheadon, Helen; Dobbin, Edwina; Ogden, Carol Anne; Dumitriu, Ingrid E.; Dunbar, Donald R.; Murray, Paul G.; Ruckerl, Dominik; Allen, Judith E.; Hume, David A.; van Rooijen, Nico; Goodlad, John R.; Freeman, Tom C.; Gregory, Christopher D.

    2015-01-01

    Summary Background Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. Results Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased “in situ transcriptomics” analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. Conclusions In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy. PMID:25702581

  3. Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal.

    PubMed

    Pletjushkina, O Yu; Fetisova, E K; Lyamzaev, K G; Ivanova, O Yu; Domnina, L V; Vyssokikh, M Yu; Pustovidko, A V; Alexeevski, A V; Alexeevski, D A; Vasiliev, J M; Murphy, M P; Chernyak, B V; Skulachev, V P

    2006-01-01

    In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis. PMID:16457620

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

    PubMed Central

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

    2015-01-01

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

  5. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells.

    PubMed

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Cho, J Hoon; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  6. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

    PubMed Central

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Hoon Cho, J; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  7. Approaches to augment CAR T-cell therapy by targeting the apoptotic machinery.

    PubMed

    Karlsson, Hannah

    2016-04-15

    Chimaeric antigen receptor (CAR) T-cells have shown impressive results in patients with B-cell leukaemia. Yet, in patients with lymphoma durable responses are still rare and heavy preconditioning required. Apoptosis resistance is considered a hallmark of cancer, often conveyed by a halted apoptosis signalling. Tumours regularly skew the balance of the components of the apoptotic machinery either through up-regulating anti-apoptotic proteins or silencing pro-apoptotic ones. Malignant B-cells frequently up-regulate anti-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins leading to therapy resistance. CAR T-cells kill tumour cells via apoptosis induction and their efficacy may be affected by the level of Bcl-2 family proteins. Hence, there is an interesting possibility to increase the effect of CAR T-cell therapy by combining it with apoptosis inhibitor blockade agents. Compounds that inhibit Bcl-2, B-cell lymphoma extra large (Bcl-xL) and Bcl-2-like protein 2 (Bcl-w), can restore execution of apoptosis in tumour cells or sensitize them to other apoptosis-dependent treatments. Hence, there is a great interest to combine such agents with CAR T-cell therapy to potentiate the effect of CAR T-cell killing. This review will focus on the potential of targeting the apoptotic machinery to sensitize tumour cells to CAR T-cell killing. PMID:27068942

  8. Loss of functional E-cadherin renders cells more resistant to the apoptotic agent taxol in vitro

    SciTech Connect

    Ferreira, Paulo; Oliveira, Maria Jose; Beraldi, Eliana; Mateus, Ana Rita; Nakajima, Takashi; Gleave, Martin; Yokota, Jun; Carneiro, Fatima; Huntsman, David; Seruca, Raquel; Suriano, Gianpaolo . E-mail: gsuriano@ipatimup.pt

    2005-10-15

    Experimental evidence supports a role for E-cadherin in suppressing invasion, metastasis, and proliferation. Germline mutations of the E-cadherin represent the genetic cause of hereditary diffuse gastric cancer (HDGC). In this type of tumor, isolated cancer cells permeate the basal membrane and paradoxically survive in the gastric wall in the absence of contact with neighbor epithelial cells or with the extracellular matrix. This suggests that upon E-cadherin deregulation, cells acquired resistance to apoptosis. To test this hypothesis, CHO cells stably expressing either wild-type E-cadherin or the HDGC-related germline mutations T340A and V832M were seeded either on a thin layer of collagen type I or on plastic and then subjected to the apoptotic agent taxol. We found that in vitro functional E-cadherin renders cells more sensitive to the effect of taxol. Our results also indicate that this effect is associated to decreased level of the anti-apoptotic bcl-2 protein.

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

    SciTech Connect

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

    1999-02-01

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

  10. Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

    SciTech Connect

    Chang, Chun-Yu; Shen, Chao-Yu; Kang, Chao-Kai; Sher, Yuh-Pyng; Sheu, Wayne H.-H.; Chang, Chia-Che; Lee, Tsung-Han

    2014-09-15

    Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. - Highlights: • Oxidized LDL induced cytotoxicity and apoptosis in HK-2 cells. • Pretreatment with taurine attenuated oxLDL-induced nephrotoxicity. • Taurine protected against renal damages through inhibition of ROS generation. • Taurine prevented apoptosis through modulation of the p53 phosphorylation.

  11. Pallbearer and friends: lending a hand in the clearance of apoptotic cells

    PubMed Central

    Elliott, Michael R.; Ravichandran, Kodi S.

    2010-01-01

    Engulfment and prompt removal of apoptotic cells occurs from embryogenesis throughout the lifespan of multi-cellular organisms. A new player, Pallbearer, has recently been identified in Drosophila as being important for efficient engulfment by macrophages. Pallbearer is a component of the SCF E3 ubiquitin ligase complex involved in ubiquitylation of proteins targeted for proteasomal degradation. This work provides the first link between the cellular processes of ubiquitylation/proteasomal degradation and the ability to efficiently clear apoptotic cells. PMID:18280734

  12. Cell-Centric View of Apoptosis and Apoptotic Cell Death-Inducing Antitumoral Strategies

    PubMed Central

    Apraiz, Aintzane; Boyano, Maria Dolores; Asumendi, Aintzane

    2011-01-01

    Programmed cell death and especially apoptotic cell death, occurs under physiological conditions and is also desirable under pathological circumstances. However, the more we learn about cellular signaling cascades, the less plausible it becomes to find restricted and well-limited signaling pathways. In this context, an extensive description of pathway-connections is necessary in order to point out the main regulatory molecules as well as to select the most appropriate therapeutic targets. On the other hand, irregularities in programmed cell death pathways often lead to tumor development and cancer-related mortality is projected to continue increasing despite the effort to develop more active and selective antitumoral compounds. In fact, tumor cell plasticity represents a major challenge in chemotherapy and improvement on anticancer therapies seems to rely on appropriate drug combinations. An overview of the current status regarding apoptotic pathways as well as available chemotherapeutic compounds provides a new perspective of possible future anticancer strategies. PMID:24212653

  13. Streptococcal pyrogenic exotoxin B inhibits apoptotic cell clearance by macrophages through protein S cleavage.

    PubMed

    Chen, Chia-Ling; Wu, Yueh-Ying; Lin, Chiou-Feng; Kuo, Chih-Feng; Han, Chia-Li; Wang, Shuying; Chuang, Woei-Jer; Chen, Chiu-Yueh; Wu, Jiunn-Jong; Tsai, Pei-Jane; Liu, Ching-Chuan; Lin, Yee-Shin

    2016-01-01

    Clearance of apoptotic cells by macrophages plays an important role in maintaining tissue homeostasis. Previous study indicated that streptococcal pyrogenic exotoxin B (SPE B) reduces phagocytic activity in group A streptococcus (GAS) infection. Here, we demonstrate that SPE B causes an inhibitory effect on protein S-mediated phagocytosis. In the presence of SPE B, serum- and purified protein S-mediated phagocytosis of apoptotic cells were significantly inhibited. The binding abilities of protein S to apoptotic cells were decreased by treatment with SPE B. Bacterial culture supernatants from GAS NZ131 strain also caused a reduction of protein S binding to apoptotic cells, but speB mutant strain did not. SPE B directly cleaved protein S in vitro and in vivo, whereas a lower level of cleavage occurred in mice infected with a speB isogenic mutant strain. SPE B-mediated initial cleavage of protein S caused a disruption of phagocytosis, and also resulted in a loss of binding ability of protein S-associated C4b-binding protein to apoptotic cells. Taken together, these results suggest a novel pathogenic role of SPE B that initiates protein S degradation followed by the inhibition of apoptotic cell clearance by macrophages. PMID:27181595

  14. Streptococcal pyrogenic exotoxin B inhibits apoptotic cell clearance by macrophages through protein S cleavage

    PubMed Central

    Chen, Chia-Ling; Wu, Yueh-Ying; Lin, Chiou-Feng; Kuo, Chih-Feng; Han, Chia-Li; Wang, Shuying; Chuang, Woei-Jer; Chen, Chiu-Yueh; Wu, Jiunn-Jong; Tsai, Pei-Jane; Liu, Ching-Chuan; Lin, Yee-Shin

    2016-01-01

    Clearance of apoptotic cells by macrophages plays an important role in maintaining tissue homeostasis. Previous study indicated that streptococcal pyrogenic exotoxin B (SPE B) reduces phagocytic activity in group A streptococcus (GAS) infection. Here, we demonstrate that SPE B causes an inhibitory effect on protein S-mediated phagocytosis. In the presence of SPE B, serum- and purified protein S-mediated phagocytosis of apoptotic cells were significantly inhibited. The binding abilities of protein S to apoptotic cells were decreased by treatment with SPE B. Bacterial culture supernatants from GAS NZ131 strain also caused a reduction of protein S binding to apoptotic cells, but speB mutant strain did not. SPE B directly cleaved protein S in vitro and in vivo, whereas a lower level of cleavage occurred in mice infected with a speB isogenic mutant strain. SPE B-mediated initial cleavage of protein S caused a disruption of phagocytosis, and also resulted in a loss of binding ability of protein S-associated C4b-binding protein to apoptotic cells. Taken together, these results suggest a novel pathogenic role of SPE B that initiates protein S degradation followed by the inhibition of apoptotic cell clearance by macrophages. PMID:27181595

  15. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    PubMed Central

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

    2014-01-01

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS).We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. PMID:25034532

  16. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells.

    PubMed

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J; Park, Daeho

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. PMID:25034532

  17. DNA typing of epithelial cells after strangulation.

    PubMed

    Wiegand, P; Kleiber, M

    1997-01-01

    DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework. PMID:9274940

  18. Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells.

    PubMed

    Park, Cheon-Soo; Ahn, Yongchel; Lee, Dahae; Moon, Sung Won; Kim, Ki Hyun; Yamabe, Noriko; Hwang, Gwi Seo; Jang, Hyuk Jai; Lee, Heesu; Kang, Ki Sung; Lee, Jae Wook

    2015-12-15

    Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24h induced cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that compound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic apoptotic pathway. PMID:26564263

  19. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  20. Overexpression of apoptotic cell removal receptor MERTK in alveolar macrophages of cigarette smokers.

    PubMed

    Kazeros, Angeliki; Harvey, Ben-Gary; Carolan, Brendan J; Vanni, Holly; Krause, Anja; Crystal, Ronald G

    2008-12-01

    Mononuclear phagocytes play an important role in the removal of apoptotic cells by expressing cell surface receptors that recognize and remove apoptotic cells. Based on the knowledge that cigarette smoking is associated with increased lung cell turnover, we hypothesized that alveolar macrophages (AMs) of normal cigarette smokers may exhibit enhanced expression of apoptotic cell removal receptor genes. AMs obtained by bronchoalveolar lavage of normal nonsmokers (n = 11) and phenotypic normal smokers (n = 13; 36 +/- 6 pack-years) were screened for mRNA expression of all known apoptotic cell removal receptors using Affymetrix HG-U133 Plus 2.0 microarray chips with TaqMan RT-PCR confirmation. Of the 14 known apoptotic receptors expressed, only MER tyrosine kinase (MERTK), a transmembrane tyrosine kinase receptor, was significantly up-regulated in smokers. MERTK expression was then assessed in AMs of smokers versus nonsmokers by TaqMan RT-PCR, immunocytochemistry, Western analysis, and flow analysis. Smoker AMs had up-regulation of MERTK mRNA levels (smoker vs. nonsmoker: 3.6-fold by microarray, P < 0.003; 9.5-fold by TaqMan RT-PCR, P < 0.02). Immunocytochemistry demonstrated a qualitative increase in MERTK protein expression on AMs of smokers. Increased protein expression of MERTK on AMs of smokers was confirmed by Western and flow analyses (P < 0.007 and P < 0.0002, respectively). MERTK, a cell surface receptor that recognizes apoptotic cells, is expressed on human AMs, and its expression is up-regulated in AMs of cigarette smokers. This up-regulation of MERTK may reflect an increased demand for removal of apoptotic cells in smokers, an observation with implications for the development of chronic obstructive pulmonary disease, a disorder associated with dysregulated apoptosis of lung parenchymal cells. PMID:18587056

  1. IMMUNE TOLERANCE INDUCTION BY APOPTOTIC CELLS REQUIRES CASPASE-DEPENDENT OXIDATION OF HMGB1

    PubMed Central

    Kazama, Hirotaka; Ricci, Jean-Ehrland; Herndon, John M.; Hoppe, George; Green, Douglas R.; Ferguson, Thomas A.

    2009-01-01

    SUMMARY The mammalian immune system discriminates between modes of cell death, with necrosis often resulting in inflammation and adaptive immunity, while apoptosis tends to be anti-inflammatory, promoting immune tolerance. In many systems immune tolerance can be established through cross presentation of antigens derived from apoptotic cells via the MHC class I pathway to CD8+ T cells. We have examined the features of apoptosis responsible for tolerance to cell-mediated immune responses in vivo, specifically the roles of caspases and the mitochondria. Our results show that caspase activation targets the mitochondria to produce ROS, which are critical to tolerance induction by apoptotic cells. ROS oxidizes the potential danger signal HMGB1 released from dying cells, thereby neutralizing its stimulatory activity and promoting tolerance. Apoptotic cells failed to induce tolerance and instead stimulated immune responses when caspase-dependent ROS activity was prohibited by scavenging or by mutation of a mitochondrial caspase target, p75 NDUSF1. Similarly blocking sites of oxidation in HMGB1 prevented tolerance induction by apoptotic cells. These results suggest that caspase orchestrated mitochondrial events determine the impact of apoptotic cells on the immune response. PMID:18631454

  2. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    PubMed Central

    Youn, Hyun-Yi; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. Results The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. Conclusions The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the

  3. Relaxin has anti-apoptotic effects on human trophoblast-derived HTR-8/SV neo cells.

    PubMed

    Lodhi, Romana S Z; Nakabayashi, Koji; Suzuki, Kaho; Yamada, Ai Y; Hazama, Rhoichi; Ebina, Yasuhiko; Yamada, Hideto

    2013-12-01

    The study was conducted to evaluate the effects of human relaxin on apoptosis in the human trophoblast derived HTR-8/SV neo cell line, which is a possible model of human extravillous trophoblasts (EVTs). HTR-8/SV neo cells, cultured in phenol red free RPMI1640 medium, were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions. RT-PCR was used for evaluating relaxin receptor: RXFP1 and RXFP2 expression in HTR-8/SV neo cells. The cell death was examined by TUNEL assay. Furthermore, we investigated caspase-3, cleaved PARP and Bcl-2 expressions by Western blot analysis to recognize the translational effects of anti-apoptotic and pro-apoptotic proteins. RXFP1 and RXFP2 mRNA expression was observed in HTR-8/SV neo cells. Compared with untreated control cultures, treatment with rH2 relaxin, decreased TUNEL-positive rate in HTR-8/SV neo cells was observed. Western blot analysis revealed that treatment with rH2 relaxin decreased the expression of caspase-3 and cleaved PARP, but in contrast increased Bcl-2 expression in those cells. These results suggest that rH2 relaxin has anti-apoptotic effects on HTR8/SV neo cells by decreasing pro-apoptotic caspase-3 and cleaved PARP expression and up-regulating anti-apoptotic Bcl-2 expression. PMID:24070111

  4. The Mannose Receptor Is Involved in the Phagocytosis of Mycobacteria-Induced Apoptotic Cells

    PubMed Central

    2016-01-01

    Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-β and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB) infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect. PMID:27413759

  5. PKC{eta} confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    SciTech Connect

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara; Livneh, Etta

    2009-09-10

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKC{eta}, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKC{eta} in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKC{eta}. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKC{eta} expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKC{eta} is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKC{eta} could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  6. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

  7. Anti-apoptotic effect of clusterin on cisplatin-induced cell death of retinoblastoma cells.

    PubMed

    Song, Hyun Beom; Jun, Hyoung-Oh; Kim, Jin Hyoung; Yu, Young Suk; Kim, Kyu-Won; Min, Bon Hong; Kim, Jeong Hun

    2013-12-01

    Clusterin is a cytoprotective chaperone protein that is known to protect various retinal cells. It was also reported to be overexpressed in several types of malignant tumors, whose chemoresistance correlates with the expression of clusterin. Herein, we investigated the effect of clusterin on cisplatin-induced cell death of retinoblastoma cells. Firstly, evaluation of clusterin expression demonstrated that it was highly expressed in human retinoblastoma tissues and cell lines (SNUOT-Rb1 and Y79) particularly in the area between viable cells around vessels and necrotic zones in the relatively avascular area in human retinoblastoma tissues. Furthermore, the effects of cisplatin on retinoblastoma cells were evaluated. Cisplatin (1 µg/ml) significantly affected cell viability of SNUOT-Rb1 cells by inducing caspase-3-dependent apoptosis. Notably, the cell death due to cisplatin was prevented by 5 µg/ml of clusterin administered 4 h prior to cisplatin treatment by inhibiting cisplatin-induced apoptosis. Furthermore, overexpression of clusterin exerted its anti-apoptotic effect on cisplatin-induced apoptosis, and effectively prevented cisplatin-induced cell death. These data suggest that clusterin, found to be expressed in human retinoblastoma, may exert anti-apoptotic effects on cisplatin-induced apoptosis and prevent cell death. Therefore, clusterin can contribute to cisplatin resistance of retinoblastoma. PMID:24085287

  8. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans. PMID:26627458

  9. Generation of Mouse Lung Epithelial Cells

    PubMed Central

    Kasinski, Andrea L.; Slack, Frank J.

    2016-01-01

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+; p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172. While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

  10. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    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. PMID:25127402

  11. Attenuation of cisplatin-induced acute renal failure is associated with less apoptotic cell death.

    PubMed

    Zhou, H; Miyaji, T; Kato, A; Fujigaki, Y; Sano, K; Hishida, A

    1999-12-01

    To clarify the pathophysiologic role of apoptosis in acute renal failure (ARF), we examined whether the attenuation of cisplatin-induced ARF is associated with the change in the degree of apoptotic cell death. The administration of cisplatin (CDDP) (6 mg/kg body weight) in rats induced ARF at day 5, as manifested by a significant increase in serum creatinine (Scr) and tubular damage. CDDP-induced apoptotic cell death was confirmed by electron microscopic examination, agarose gel electrophoresis, and increased cells positive for TaT-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) in the outer medulla of the kidney. Treatment with dimethylthiourea (DMTU)--a scavenger of hydroxyl radicals--or glycine abrogated CDDP-induced increases in Scr, the tubular damage score, and the number of TUNEL-positive cells. Pretreatment with uranyl acetate (UA) induced a significant expression of Bcl-2 in the kidney and ameliorated CDDP-induced increases in Scr, the tubular damage score, and TUNEL-positive cells in the outer stripe of the outer medulla. Our findings indicate (1) that the attenuation of CDDP-induced ARF was associated with less apoptotic cell death and (2) that the induction of the anti-apoptotic protein Bcl-2 attenuated apoptosis and tubular damage. Our results suggest that apoptotic cell death may play an important role in the development of cisplatin-induced ARF. PMID:10595794

  12. Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Brauchle, Eva; Thude, Sibylle; Brucker, Sara Y.; Schenke-Layland, Katja

    2014-04-01

    Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes.

  13. Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy

    PubMed Central

    Brauchle, Eva; Thude, Sibylle; Brucker, Sara Y.; Schenke-Layland, Katja

    2014-01-01

    Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes. PMID:24732136

  14. Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways

    PubMed Central

    YUAN, HONGYAN; WANG, JUAN; WANG, FENGLI; ZHANG, NANNAN; LI, QIONGSHU; XIE, FEI; CHEN, TANXIU; ZHAI, RUIPING; WANG, FANG; GUO, YINGYING; NI, WEIHUA; TAI, GUIXIANG

    2015-01-01

    Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC-7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC-7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC-7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase-8 and caspase-9 were activated in MUC1 gene-silenced SMMC-7721 cells. The pro-apoptotic protein Bcl-2-associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti-apoptotic protein B-cell lymphoma 2 was decreased in MUC1 gene-silenced cells. In addition, results from the co-immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase-8 and these interactions were reduced upon MUC1 gene silencing in SMMC-7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC-7721 cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways. PMID:26398332

  15. Assessment of the Cytotoxic and Apoptotic Effects of Chaetominine in a Human Leukemia Cell Line

    PubMed Central

    Yao, Jingyun; Jiao, Ruihua; Liu, Changqing; Zhang, Yupeng; Yu, Wanguo; Lu, Yanhua; Tan, Renxiang

    2016-01-01

    Chaetominine is a quinazoline alkaloid originating from the endophytic fungus Aspergillus fumigatus CY018. In this study, we showed evidence that chaetominine has cytotoxic and apoptotic effects on human leukemia K562 cells and investigated the pathway involved in chaetominine-induced apoptosis in detail. Chaetominine inhibited K562 cell growth, with an IC50 value of 35 nM, but showed little inhibitory effect on the growth of human peripheral blood mononuclear cells. The high apoptosis rates, morphological apoptotic features, and DNA fragmentation caused by chaetominine indicated that the cytotoxicity was partially caused by its pro-apoptotic effect. Under chaetominine treatment, the Bax/Bcl-2 ratio was upregulated (from 0.3 to 8), which was followed by a decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, and stimulation of Apaf-1. Furthermore, activation of caspase-9 and caspase-3, which are the main executers of the apoptotic process, was observed. These results demonstrated that chaetominine induced cell apoptosis via the mitochondrial pathway. Chaetominine inhibited K562 cell growth and induced apoptotic cell death through the intrinsic pathway, which suggests that chaetominine might be a promising therapeutic for leukemia. PMID:26902083

  16. Cyclosporine A induces apoptotic and autophagic cell death in rat pituitary GH3 cells.

    PubMed

    Kim, Han Sung; Choi, Seung-Il; Jeung, Eui-Bae; Yoo, Yeong-Min

    2014-01-01

    Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels. PMID:25299210

  17. Selective cytotoxicity and modulation of apoptotic signature of breast cancer cells by Pithecellobium dulce leaf extracts.

    PubMed

    Sharma, Monika

    2016-05-01

    We report the potent and selective cytotoxicity of the crude aqueous leaf extract from the medicinal plant, Pithecellobium dulce toward the human breast cancer cells (MCF-7), but not the normal cells (MCF-10A). The cytotoxicity was found to be dose and time dependent, as 300 µg/mL of the extract decreased the cell viability to 50% (IC50 ) in 48 h. The induction of apoptosis in the breast cancer cells after treatment was confirmed by significant percentage (24.7%), of early apoptotic cells (AnnexinV (+) Propidium Iodide(_) ) in treated cells as compared to control cells (3.5%). We observed a significant upregulation in the mRNA expression of various pro-apoptotic gene such as Bax (21.1 folds), p21(14.4 folds), p53 (11.7 folds), TNF (10.2 folds) and fas (6.3 folds) after treatment as compared to untreated cells. On the other hand, the relative mRNA expression of anti-apoptotic genes such as Bcl-2, NF-KB and Cdk was reduced. The selective upregulation of pro-apoptotic gene and down regulation of specific anti-apoptotic genes could be the inducing factor for apoptotic cell death in MCF-7 cells after treatment with the herbal extract. We believe that our findings provide a foundation for further studies on this formulation as a potential therapeutic candidate for breast cancer. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:756-766, 2016. PMID:26996293

  18. GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

    PubMed

    Reshi, Latif; Wu, Horng-Cherng; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-04-01

    Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these

  19. Selective apoptotic effects of piceatannol and myricetin in human cancer cells.

    PubMed

    Morales, Paloma; Haza, Ana I

    2012-12-01

    Numerous studies have shown the potential of dietary polyphenols as anticarcinogenic agents. The aim of the present study was to evaluate the apoptotic effects of piceatannol and myricetin, naturally occurring polyphenols in red wine, alone or in combination, in two human cell lines: HL-60 (leukemia) and HepG2 (hepatoma). Apoptotic cells were identified by chromatin condensation, poly(ADP-ribose) polymerase cleavage and flow cytometry analysis. Results from TUNEL assay showed that piceatannol or myricetin alone induced apoptotic cell death in a concentration- and time-dependent manners in HL-60 cells. Furthermore, in combined treatment the percentage of apoptotic HL-60 cells was significantly higher. Nevertheless, the percentage of TUNEL positive HepG2 cells only was significant after piceatannol treatment and in combined treatment was even lower than in cells treated with piceatannol alone. Moreover, we also studied the relative reactive oxygen species (ROS) production. Our results indicate that apoptosis induced by piceatannol or myricetin occurs through an ROS-independent cell death pathway. In conclusion, piceatannol and myricetin synergistically induced apoptosis in HL-60 cells but not in HepG2 cells. These findings suggest that the potential anticarcinogenic properties of dietary polyphenols depend largely on the cell line used. The relevance of these data needs to be verified in human epidemiological studies. PMID:21935971

  20. Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages

    NASA Astrophysics Data System (ADS)

    Freire-de-Lima, Célio G.; Nascimento, Danielle O.; Soares, Milena B. P.; Bozza, Patricia T.; Castro-Faria-Neto, Hugo C.; de Mello, Fernando G.; Dosreis, George A.; Lopes, Marcela F.

    2000-01-01

    After apoptosis, phagocytes prevent inflammation and tissue damage by the uptake and removal of dead cells. In addition, apoptotic cells evoke an anti-inflammatory response through macrophages. We have previously shown that there is intense lymphocyte apoptosis in an experimental model of Chagas' disease, a debilitating cardiac illness caused by the protozoan Trypanosoma cruzi. Here we show that the interaction of apoptotic, but not necrotic T lymphocytes with macrophages infected with T. cruzi fuels parasite growth in a manner dependent on prostaglandins, transforming growth factor-β (TGF-β) and polyamine biosynthesis. We show that the vitronectin receptor is critical, in both apoptotic-cell cytoadherence and the induction of prostaglandin E2/TGF-β release and ornithine decarboxylase activity in macrophages. A single injection of apoptotic cells in infected mice increases parasitaemia, whereas treatment with cyclooxygenase inhibitors almost completely ablates it in vivo. These results suggest that continual lymphocyte apoptosis and phagocytosis of apoptotic cells by macrophages have a role in parasite persistence in the host, and that cyclooxygenase inhibitors have potential therapeutic application in the control of parasite replication and spread in Chagas' disease.

  1. Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

    PubMed Central

    Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

    2014-01-01

    Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001 PMID:24668173

  2. Vessel-associated myogenic precursors control macrophage activation and clearance of apoptotic cells.

    PubMed

    Bosurgi, L; Brunelli, S; Rigamonti, E; Monno, A; Manfredi, A A; Rovere-Querini, P

    2015-01-01

    Swift and regulated clearance of apoptotic cells prevents the accumulation of cell remnants in injured tissues and contributes to the shift of macrophages towards alternatively activated reparatory cells that sustain wound healing. Environmental signals, most of which are unknown, in turn control the efficiency of the clearance of apoptotic cells and as such determine whether tissues eventually heal. In this study we show that vessel-associated stem cells (mesoangioblasts) specifically modulate the expression of genes involved in the clearance of apoptotic cells and in macrophage alternative activation, including those of scavenger receptors and of molecules that bridge dying cells and phagocytes. Mesoangioblasts, but not immortalized myoblasts or neural precursor cells, enhance CD163 membrane expression in vitro as assessed by flow cytometry, indicating that the effect is specific. Mesoangioblasts transplanted in acutely or chronically injured skeletal muscles determine the expansion of the population of CD163(+) infiltrating macrophages and increase the extent of CD163 expression. Conversely, macrophages challenged with mesoangioblasts engulf significantly better apoptotic cells in vitro. Collectively, the data reveal a feed-forward loop between macrophages and vessel-associated stem cells, which has implications for the skeletal muscle homeostatic response to sterile injury and for diseases in which homeostasis is jeopardized, including muscle dystrophies and inflammatory myopathies. PMID:24749786

  3. Unexpected requirement for ELMO1 in clearance of apoptotic germ cells in vivo.

    PubMed

    Elliott, Michael R; Zheng, Shuqiu; Park, Daeho; Woodson, Robin I; Reardon, Michael A; Juncadella, Ignacio J; Kinchen, Jason M; Zhang, Jun; Lysiak, Jeffrey J; Ravichandran, Kodi S

    2010-09-16

    Apoptosis and the subsequent clearance of dying cells occurs throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases. ELMO1 is an evolutionarily conserved cytoplasmic engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with DOCK1 and the GTPase RAC1, promotes internalization of the dying cells. Here we report the generation of ELMO1-deficient mice, which we found to be unexpectedly viable and grossly normal. However, they had a striking testicular pathology, with disrupted seminiferous epithelium, multinucleated giant cells, uncleared apoptotic germ cells and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The engulfment receptor BAI1 and RAC1 (upstream and downstream of ELMO1, respectively) were also important for Sertoli-cell-mediated engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli-cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between engulfment and tissue homeostasis in vivo. PMID:20844538

  4. Caco-2 cells infected with rotavirus release extracellular vesicles that express markers of apoptotic bodies and exosomes.

    PubMed

    Bautista, Diana; Rodríguez, Luz-Stella; Franco, Manuel A; Angel, Juana; Barreto, Alfonso

    2015-07-01

    Previously, we showed that infecting human intestinal epithelial cells (Caco-2) with rotavirus (RV) increases the release of extracellular vesicles (EVs) with an immunomodulatory function that, upon concentration at 100,000×g, present buoyant densities on a sucrose gradient of between 1.10 to 1.18 g/ml (characteristic of exosomes) and higher than 1.24 g/ml (proposed for apoptotic bodies). The effect of cellular death induced by RV on the composition of these EV is unknown. Here, we evaluated exosome (CD63, Hsc70, and AChE) and apoptotic body (histone H3) markers in EVs isolated by differential centrifugation (4000×g, 10,000×g, and 100,000×g) or filtration/ultracentrifugation (100,000×g) protocols. When we infected cells in the presence of caspase inhibitors, Hsc70 and AChE diminished in EVs obtained at 100,000×g, but not in EVs obtained at 4000×g or 10,000×g. In addition, caspase inhibitors decreased CD63 and AChE in vesicles with low and high buoyant densities. Without caspase inhibitors, RV infection increased exosome markers in all of the EVs obtained by differential centrifugation. However, CD63 preferentially localized in the 100,000×g fraction and H3 only increased in EVs concentrated at 100,000×g and with high buoyant densities on a sucrose gradient. Thus, RV infection increases the release of EVs that, upon concentration at 100,000×g, are composed by exosomes and apoptotic bodies, which can partially be separated using sucrose gradients. PMID:25975376

  5. Pro‑apoptotic effects of pycnogenol on HT1080 human fibrosarcoma cells.

    PubMed

    Harati, Kamran; Slodnik, Pawel; Chromik, Ansgar Michael; Behr, Björn; Goertz, Ole; Hirsch, Tobias; Kapalschinski, Nicolai; Klein-Hitpass, Ludger; Kolbenschlag, Jonas; Uhl, Waldemar; Lehnhardt, Marcus; Daigeler, Adrien

    2015-04-01

    Complete surgical resection with clear margins remains the mainstay of therapy for localised fibrosarcomas. Nevertheless, metastatic fibrosarcomas still represent a therapeutic dilemma. Commonly used chemotherapeutic agents like doxorubicin have proven to be effective in <30% of all cases of disseminated fibrosarcoma. Especially elderly patients with cardiac subdisease are not suitable for systemic chemotherapy with doxorubicin. Therefore we tested the apoptotic effects of the well-tolerated pine bark extract pycnogenol and its constituents on human fibrosarcoma cells (HT1080). Ten healthy subjects (six females, four males, mean age 24.8 ± 6 years) received a single dose of 300 mg pycnogenol orally. Blood plasma samples were obtained before and 6 h after intake of pycnogenol. HT1080 cells were treated with these plasma samples. Additionally, HT1080 were incubated separately with catechin, epicatechin and taxifolin that are known as the main constituents of pycnogenol. Vital, apoptotic and necrotic cells were quantified using flow cytometric analysis. Gene expression was analyzed by RNA microarray. The results showed that single application of taxifolin, catechin and epicatechin reduced cell viability of HT1080 cells only moderately. A single dose of 300 mg pycnogenol given to 10 healthy adults produced plasma samples that led to significant apoptotic cell death ex vivo whereas pycnogenol-negative serum displayed no apoptotic activity. Microarray analysis revealed remarkable expression changes induced by pycnogenol in a variety of genes, which are involved in different apoptotic pathways of cancer cells [Janus kinase 1 (JAK1), DUSP1, RHOA, laminin γ1 (LAMC1), fibronectin 1 (FN1), catenin α1 (CTNNA1), ITGB1]. In conclusion, metabolised pycnogenol induces apoptosis in human fibrosarcoma cells. Pycnogenol exhibits its pro-apoptotic activity as a mixture and is more effective than its main constituents catechin, epicatechin and taxifolin indicating that the

  6. Natural compound Alternol induces oxidative stress-dependent apoptotic cell death preferentially in prostate cancer cells

    PubMed Central

    Tang, Yuzhe; Chen, Ruibao; Huang, Yan; Li, Guodong; Huang, Yiling; Chen, Jiepeng; Duan, Lili; Zhu, Bao-Ting; Thrasher, J Brantley; Zhang, Xu; Li, Benyi

    2014-01-01

    Prostate cancers at the late stage of castration resistance are not responding well to most of current therapies available in clinic, reflecting a desperate need of novel treatment for this life-threatening disease. In this study, we evaluated the anti-cancer effect of a recently isolated natural compound Alternol in multiple prostate cancer cell lines with the properties of advanced prostate cancers in comparison to prostate-derived non-malignant cells. As assessed by trypan blue exclusion assay, a significant cell death was observed in all prostate cancer cell lines except DU145 but not in non-malignant (RWPE-1and BPH1) cells. Further analyses revealed that Alternol-induced cell death was an apoptotic response in a dose- and time-dependent manner, as evidenced by the appearance of apoptosis hallmarks such as Caspase-3 processing and PARP cleavage. Interestingly, Alternol-induced cell death was completely abolished by reactive oxygen species (ROS) scavengers, N-acetylcysteine (N-Ac) and dihydrolipoic acid (DHLA). We also demonstrated that the pro-apoptotic Bax protein was activated after Alternol treatment and was critical for Alternol-induced apoptosis. Animal xenograft experiments in nude mice showed that Alternol treatment largely suppressed tumor growth of PC-3 xenografts but not Bax-null DU-145 xenografts in vivo. These data suggest that Alternol might serve as a novel anticancer agent for late stage prostate cancer patient. PMID:24688053

  7. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.

    PubMed

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  8. Formation of a Neurosensory Organ by Epithelial Cell Slithering.

    PubMed

    Kuo, Christin S; Krasnow, Mark A

    2015-10-01

    Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic. PMID:26435104

  9. Caspase dependent apoptotic inhibition of melanoma and lung cancer cells by tropical Rubus extracts.

    PubMed

    George, Blassan Plackal Adimuriyil; Abrahamse, Heidi; Hemmaragala, Nanjundaswamy M

    2016-05-01

    Rubus fairholmianus Gard. inhibits human melanoma (A375) and lung cancer (A549) cell growth by the caspase dependent apoptotic pathway. Herbal products have a long history of clinical use and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. The plants and plant derived products became the basis of traditional medicine system throughout the world for thousands of years. The effects of R. fairholmianus root acetone extract (RFRA) on the proliferation of A375 and A549 cells was examined in this study. RFRA led to a decrease in cell viability, proliferation and an increase in cytotoxicity in a dose dependent manner when compared with control and normal skin fibroblast cells (WS1). The morphology of treated cells supported apoptotic cell death. Annexin V/propidium iodide staining indicated that RFRA induced apoptosis in A375 and A549 cells and the percentages of early and late apoptotic populations significantly increased. Moreover, the apoptotic inducing ability of RFRA when analysing effector caspase 3/7 activity, indicated a marked increase in treated cells. In summary, we have shown the anticancer effects of RFRA in A375 and A549 cancer cells via induction of caspase dependent apoptosis in vitro. The extract is more effective against melanoma; which may suggest the usefulness of RFRA-based anticancer therapies. PMID:27133056

  10. Ouabain modulates ciliogenesis in epithelial cells

    PubMed Central

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

    2011-01-01

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

  11. Is the inflammasome relevant for epithelial cell function?

    PubMed

    Santana, Patricia T; Martel, Jan; Lai, Hsin-Chih; Perfettini, Jean-Luc; Kanellopoulos, Jean M; Young, John D; Coutinho-Silva, Robson; Ojcius, David M

    2016-02-01

    Inflammasomes are intracellular protein complexes that sense microbial components and damage of infected cells. Following activation by molecules released by pathogens or injured cells, inflammasomes activate caspase-1, allowing secretion of the pro-inflammatory cytokines IL-1β and IL-18 from innate immune cells. Inflammasomes are also expressed in epithelial cells, where their function has attracted less attention. Nonetheless, depending on the tissue, epithelial inflammasomes can mediate inflammation, wound healing, and pain sensitivity. We review here recent findings on inflammasomes found in epithelial tissues, highlighting the importance of these protein complexes in the response of epithelial tissues to microbial infections. PMID:26546965

  12. Calcium oxalate toxicity in renal epithelial cells: the mediation of crystal size on cell death mode

    PubMed Central

    Sun, X-Y; Gan, Q-Z; Ouyang, J-M

    2015-01-01

    The cytotoxicity of calcium oxalate (CaOx) in renal epithelial cells has been studied extensively, but the cell death mode induced by CaOx with different physical properties, such as crystal size and crystal phase, has not been studied in detail. In this study, we comparatively investigated the differences of cell death mode induced by nano-sized (50 nm) and micron-sized (10 μm) calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) to explore the cell death mechanism. The effect of the exposure of nano-/micron-sized COM and COD crystals toward the African green monkey renal epithelial (Vero) cells were investigated by detecting cell cytoskeleton changes, lysosomal integrity, mitochondrial membrane potential (Δψm), apoptosis and/or necrosis, osteopontin (OPN) expression, and malondialdehyde (MDA) release. Nano-/micron-sized COM and COD crystals could cause apoptosis and necrosis simultaneously. Nano-sized crystals primarily caused apoptotic cell death, leading to cell shrinkage, phosphatidylserine ectropion, and nuclear shrinkage, whereas micron-sized crystals primarily caused necrotic cell death, leading to cell swelling and cell membrane and lysosome rupture. Nano-sized COM and COD crystals induced much greater cell death (sum of apoptosis and necrosis) than micron-sized crystals, and COM crystals showed higher cytotoxicity than the same-sized COD crystals. Both apoptosis and necrosis could lead to mitochondria depolarization and elevate the expression of OPN and the generation of lipid peroxidation product MDA. The amount of expressed OPN and generated MDA was positively related to cell injury degree. The physicochemical properties of crystals could affect the cell death mode. The results of this study may provide a basis for future studies on cell death mechanisms. PMID:27551481

  13. Apoptotic and autophagic cell death induced by glucolaxogenin in cervical cancer cells.

    PubMed

    Sánchez-Sánchez, L; Escobar, M L; Sandoval-Ramírez, J; López-Muñoz, H; Fernández-Herrera, M A; Hernández-Vázquez, J M V; Hilario-Martínez, C; Zenteno, E

    2015-12-01

    The antiproliferative and cytotoxic activity of glucolaxogenin and its ability to induce apoptosis and autophagy in cervical cancer cells are reported. We ascertained that glucolaxogenin exerts an inhibitory effect on the proliferation of HeLa, CaSki and ViBo cells in a dose-dependent manner. Analysis of DNA distribution in the cell-cycle phase of tumor cells treated with glucolaxogenin suggests that the anti-proliferative activity of this steroid is not always dependent on the cell cycle. Cytotoxic activity was evaluated by detection of the lactate dehydrogenase enzyme in supernatants from tumor cell cultures treated with the steroid. Glucolaxogenin exhibited null cytotoxic activity. With respect to the apoptotic activity, the generation of apoptotic bodies, the presence of active caspase-3 and annexin-V, as well as the DNA fragmentation observed in all tumor lines after treatment with glucolaxogenin suggests that this compound does indeed induce cell death by apoptosis. Also, a significantly increased presence of the LC3-II, LC3 and Lamp-1 proteins was evidenced with the ultrastructural existence of autophagic vacuoles in cells treated with this steroidal glycoside, indicating that glucolaxogenin also induces autophagic cell death. It is important to note that this compound showed no cytotoxic effect and did not affect the proliferative capacity of mononuclear cells obtained from normal human peripheral blood activated by phytohaemagglutinin. Thus, glucolaxogenin is a compound with anti-proliferative properties that induces programmed cell death in cancer cell lines, though it is selective with respect to normal lymphocytic cells. These findings indicate that this glycoside could have a selective action on tumor cells and, therefore, be worthy of consideration as a therapeutic candidate with anti-tumor potential. PMID:26437916

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

    PubMed Central

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

    2016-01-01

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

  15. Anti-apoptotic peptides protect against radiation-induced cell death

    SciTech Connect

    McConnell, Kevin W.; Muenzer, Jared T.; Chang, Kathy C.; Davis, Chris G.; McDunn, Jonathan E.; Coopersmith, Craig M.; Hilliard, Carolyn A.; Hotchkiss, Richard S.; Grigsby, Perry W.; Hunt, Clayton R. . E-mail: chunt@radonc.wustl.edu

    2007-04-06

    The risk of terrorist attacks utilizing either nuclear or radiological weapons has raised concerns about the current lack of effective radioprotectants. Here it is demonstrated that the BH4 peptide domain of the anti-apoptotic protein Bcl-xL can be delivered to cells by covalent attachment to the TAT peptide transduction domain (TAT-BH4) and provide protection in vitro and in vivo from radiation-induced apoptotic cell death. Isolated human lymphocytes treated with TAT-BH4 were protected against apoptosis following exposure to 15 Gy radiation. In mice exposed to 5 Gy radiation, TAT-BH4 treatment protected splenocytes and thymocytes from radiation-induced apoptotic cell death. Most importantly, in vivo radiation protection was observed in mice whether TAT-BH4 treatment was given prior to or after irradiation. Thus, by targeting steps within the apoptosis signaling pathway it is possible to develop post-exposure treatments to protect radio-sensitive tissues.

  16. Establishment of Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium.

    PubMed

    Nam, Hyun; Kim, Ji-Hye; Kim, Jae-Won; Seo, Byoung-Moo; Park, Joo-Cheol; Kim, Jung-Wook; Lee, Gene

    2014-07-01

    Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth. PMID:25081036

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

    SciTech Connect

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

    2004-07-14

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

  18. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

    PubMed Central

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

    2016-01-01

    Summary Cellular senescence suppresses cancer by arresting cells at risk of 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. Furthermore, 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. PMID:15657080

  19. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

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

    2013-01-01

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

  20. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

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

    2013-03-01

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

  1. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

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

  2. Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions

    PubMed Central

    2009-01-01

    Background Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. Results EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. Conclusion This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions. PMID:19883504

  3. Bim mediates mitochondria-regulated particulate matter-induced apoptosis in alveolar epithelial cells

    PubMed Central

    Zhang, J.; Ghio, A.J.; Chang, W.; Kamdar, O.; Rosen, G.D.; Upadhyay, D.

    2007-01-01

    We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 μm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway. PMID:17716672

  4. Different death stimuli evoke apoptosis via multiple pathways in retinal pigment epithelial cells.

    PubMed

    Ferrington, Deborah A; Tran, Tina N; Lew, Kathleen L; Van Remmen, Holly; Gregerson, Dale S

    2006-09-01

    Loss of retinal pigment epithelial (RPE) cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration, and with light damage. Strategies for preservation of vision that would interrupt the apoptotic cascade require understanding the molecular events associated with apoptosis. This study investigated the susceptibility of RPE to caspase-dependent and -independent apoptotic pathways when challenged with different stimuli, including oxidants, anti-Fas antibody, and activated cytotoxic T lymphocytes (CTLs). These experiments used novel RPE cell lines developed from wildtype and heterozygous mice with reduced levels of either Mn superoxide dismutatse (SOD) or CuZnSOD. Peroxide and 4-hydroxynonenal induced apoptosis through both caspase-independent and -dependent pathways, respectively. With both oxidants, translocation of apoptosis inducing factor into the nucleus was observed. Cells containing reduced levels of CuZnSOD were the most susceptible to oxidant-induced cell death. Targeted killing by CTLs and activation of the Fas death receptor induced caspase-dependent apoptosis. These results show stimulus-specific activation of either the caspase-dependent or -independent pathway. Since cultured RPE express the protein components required for different apoptotic pathways, they provide a good model system for studying molecular events associated with multiple signals that lead to cell death. PMID:16682026

  5. Recognition of apoptotic cells by viable cells is specific, ubiquitous, and species independent: analysis using photonic crystal biosensors

    PubMed Central

    Pattabiraman, Goutham; Lidstone, Erich A.; Palasiewicz, Karol; Cunningham, Brian T.; Ucker, David S.

    2014-01-01

    Apoptotic recognition is innate and linked to a profound immune regulation (innate apoptotic immunity [IAI]) involving anti-inflammatory and immunosuppressive responses. Many of the molecular and mechanistic details of this response remain elusive. Although immune outcomes can be quantified readily, the initial specific recognition events have been difficult to assess. We developed a sensitive, real-time method to detect the recognition of apoptotic cells by viable adherent responder cells, using a photonic crystal biosensor approach. The method relies on characteristic spectral shifts resulting from the specific recognition and dose-dependent interaction of adherent responder cells with nonadherent apoptotic targets. Of note, the biosensor provides a readout of early recognition-specific events in responder cells that occur distal to the biosensor surface. We find that innate apoptotic cell recognition occurs in a strikingly species-independent manner, consistent with our previous work and inferences drawn from indirect assays. Our studies indicate obligate cytoskeletal involvement, although apoptotic cell phagocytosis is not involved. Because it is a direct, objective, and quantitative readout of recognition exclusively, this biosensor approach affords a methodology with which to dissect the early recognition events associated with IAI and immunosuppression. PMID:24694594

  6. Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function.

    PubMed

    Ma, S B; Nguyen, T N; Tan, I; Ninnis, R; Iyer, S; Stroud, D A; Menard, M; Kluck, R M; Ryan, M T; Dewson, G

    2014-12-01

    In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-xL. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family

  7. Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

    PubMed Central

    Ma, S B; Nguyen, T N; Tan, I; Ninnis, R; Iyer, S; Stroud, D A; Menard, M; Kluck, R M; Ryan, M T; Dewson, G

    2014-01-01

    In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-xL. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family

  8. Rapid reuptake of granzyme B leads to emperitosis: an apoptotic cell-in-cell death of immune killer cells inside tumor cells.

    PubMed

    Wang, S; He, M-f; Chen, Y-h; Wang, M-y; Yu, X-M; Bai, J; Zhu, H-y; Wang, Y-y; Zhao, H; Mei, Q; Nie, J; Ma, J; Wang, J-f; Wen, Q; Ma, L; Wang, Y; Wang, X-n

    2013-01-01

    A cell-in-cell process refers to the invasion of one living cell into another homotypic or heterotypic cell. Different from non-apoptotic death processes of internalized cells termed entosis or cannibalism, we previously reported an apoptotic cell-in-cell death occurring during heterotypic cell-in-cell formation. In this study, we further demonstrated that the apoptotic cell-in-cell death occurred only in internalized immune killer cells expressing granzyme B (GzmB). Vacuole wrapping around the internalized cells inside the target cells was the common hallmark during the early stage of all cell-in-cell processes, which resulted in the accumulation of reactive oxygen species and subsequent mitochondrial injury of encapsulated killer or non-cytotoxic immune cells. However, internalized killer cells mediated rapid bubbling of the vacuoles with the subsequent degranulation of GzmB inside the vacuole of the target cells and underwent the reuptake of GzmB by killer cells themselves. The confinement of GzmB inside the vacuole surpassed the lysosome-mediated cell death occurring in heterotypic or homotypic entosis processes, resulting in a GzmB-triggered caspase-dependent apoptotic cell-in-cell death of internalized killer cells. On the contrary, internalized killer cells from GzmB-deficient mice underwent a typical non-apoptotic entotic cell-in-cell death similar to that of non-cytotoxic immune cells or tumor cells. Our results thus demonstrated the critical involvement of immune cells with cytotoxic property in apoptotic cell-in-cell death, which we termed as emperitosis taken from emperipolesis and apoptosis. Whereas entosis or cannibalism may serve as a feed-on mechanism to exacerbate and nourish tumor cells, emperitosis of immune killer cells inside tumor cells may serve as an in-cell danger sensation model to prevent the killing of target cells from inside, implying a unique mechanism for tumor cells to escape from immune surveillance. PMID:24113190

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

    PubMed Central

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

    2012-01-01

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

  10. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  11. Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-of-Function Capacity*

    PubMed Central

    Nguyen, Khanh-Quynh N.; Tsou, Wen-I; Calarese, Daniel A.; Kimani, Stanley G.; Singh, Sukhwinder; Hsieh, Shelly; Liu, Yongzhang; Lu, Bin; Wu, Yi; Garforth, Scott J.; Almo, Steve C.; Kotenko, Sergei V.; Birge, Raymond B.

    2014-01-01

    MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells. PMID:25074939

  12. The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis.

    PubMed

    Wijeyesakere, Sanjeeva Joseph; Bedi, Sukhmani Kaur; Huynh, David; Raghavan, Malini

    2016-05-01

    Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum. Calreticulin is detectable on the surface of apoptotic cells under some apoptosis-inducing conditions, where it promotes the phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains: a globular domain, an extended arm-like P-domain, and a C-terminal acidic region containing multiple low-affinity calcium binding sites. We show that calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared with other phospholipids and that this interaction is calcium dependent. Additionally, exogenous calreticulin binds apoptotic cells via a higher-affinity calcium-dependent mode that is acidic region dependent. Exogenous calreticulin also binds live cells, including macrophages, via a second, lower-affinity P-domain and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide binding site. Truncation constructs lacking the acidic region or arm-like P-domain of calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid binding site of calreticulin as a key anchor point for the cell surface expression of calreticulin on apoptotic cells. These findings also support a role for calreticulin as a PS-bridging molecule that cooperates with other PS-binding factors to promote the phagocytosis of apoptotic cells. PMID:27036911

  13. Purified Essential Oil from Ocimum sanctum Linn. Triggers the Apoptotic Mechanism in Human Breast Cancer Cells

    PubMed Central

    Manaharan, Thamilvaani; Thirugnanasampandan, Ramaraj; Jayakumar, Rajarajeswaran; Kanthimathi, M. S.; Ramya, Gunasekar; Ramnath, Madhusudhanan Gogul

    2016-01-01

    Background: Essential oil of Ocimum sanctum Linn. exhibited various pharmacological activities including antifungal and antimicrobial activities. In this study, we analyzed the anticancer and apoptosis mechanisms of Ocimum sanctum essential oil (OSEO). Objective: To trigger the apoptosis mechanism in human breast cancer cells using OSEO. Materials and Methods: OSEO was extracted using hydrodistillation of the leaves. Cell proliferation was determined using different concentrations of OSEO. Apoptosis studies were carried out in human breast cancer cells using propidium iodide (PI) and Hoechst staining. Results: We found that OSEO inhibited proliferation (IC50 = 170 μg/ml) of Michigan cancer foundation-7 (MCF-7) cells in a dose-dependent manner. The OSEO also induced apoptosis as evidenced by the increasing number of PI-stained apoptotic nucleic of MCF-7 cells. Flow cytometry analysis revealed that treatment with OSEO (50–500 μg/ml) increased the apoptotic cells population (16–84%) dose dependently compared to the control. OSEO has the ability to up-regulate the apoptotic genes p53 and Bid and as well as elevates the ratio of Bax/Bcl-2. Conclusion: Our findings indicate that OSEO has the ability as proapoptotic inducer and it could be developed as an anticancer agent. SUMMARY OSEO inhibited proliferation of MCF-7 cells with an IC50 of 170 μg/mLOSEO at 500 μg/mL increased the population of apoptotic cells by 84%OSEO up-regulated the expression of apoptotic genes and as well increased the Bax/Bcl2 ratio. Abbreviations used: BAX: BAX BCL2-associated X protein; BCL2: B-cell CLL/lymphoma 2; BID: BH3 Interacting domain death agonist; OSEO: Ocimum sanctum essential oil; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco's modified Eagle medium; MCF-7: Michigan cancer foundation-7; RT-PCR: Real Time Polymerase Chain Reaction.

  14. Applications of mouse airway epithelial cell culture for asthma research.

    PubMed

    Horani, Amjad; Dickinson, John D; Brody, Steven L

    2013-01-01

    Primary airway epithelial cell culture provides a valuable tool for studying cell differentiation, cell-cell interactions, and the role of immune system factors in asthma pathogenesis. In this chapter, we discuss the application of mouse tracheal epithelial cell cultures for the study of asthma biology. A major advantage of this system is the ability to use airway epithelial cells from mice with defined genetic backgrounds. The in vitro proliferation and differentiation of mouse airway epithelial cells uses the air-liquid interface condition to generate well-differentiated epithelia with characteristics of native airways. Protocols are provided for manipulation of differentiation, induction of mucous cell metaplasia, genetic modification, and cell and pathogen coculture. Assays for the assessment of gene expression, responses of cells, and analysis of specific cell subpopulations within the airway epithelium are included. PMID:23943446

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

    PubMed Central

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

    2016-01-01

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

  16. Epithelial in vitro cell systems in carcinogenesis studies

    SciTech Connect

    Borek, C.

    1983-01-01

    The development of epithelial cells systems to study oncogenic transformation has presented a major challenge in the field of carcinogenesis. Because there exists in man a preponderance of carcinomas over sarcomas, the importance of studying oncogenic transformation in epithelial cells is of great relevance to human disease. The difficulty lies in the fact that different tissues contain epithelial cells with singular differentiated characteristics, which must be defined to assert the different nature of the cells being used. Liver cells in culture are a case in point. By careful maintenance and optimal culture conditions, one can maintain many of the differentiated characteristics of the cells for prolonged periods of time.

  17. How does ethanol induce apoptotic cell death of SK-N-SH neuroblastoma cells.

    PubMed

    Moon, Yong; Kwon, Yongil; Yu, Shun

    2013-07-15

    A body of evidence suggests that ethanol can lead to damage of neuronal cells. However, the mechanism underlying the ethanol-induced damage of neuronal cells remains unclear. The role of mitogen-activated protein kinases in ethanol-induced damage was investigated in SK-N-SH neuroblastoma cells. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide cell viability assay, DNA fragmentation detection, and flow cytometric analysis showed that ethanol induced apoptotic cell death and cell cycle arrest, characterized by increased caspase-3 activity, DNA fragmentation, nuclear disruption, and G1 arrest of cell cycle of the SK-N-SH neuroblastoma cells. In addition, western blot analysis indicated that ethanol induced a lasting increase in c-Jun N-terminal protein kinase activity and a transient increase in p38 kinase activity of the neuroblastoma cells. c-Jun N-terminal protein kinase or p38 kinase inhibitors significantly reduced the ethanol-induced cell death. Ethanol also increased p53 phosphorylation, followed by an increase in p21 tumor suppressor protein and a decrease in phospho-Rb (retinoblastoma) protein, leading to alterations in the expressions and activity of cyclin dependent protein kinases. Our results suggest that ethanol mediates apoptosis of SK-N-SH neuroblastoma cells by activating p53-related cell cycle arrest possibly through activation of the c-Jun N-terminal protein kinase-related cell death pathway. PMID:25206494

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

    PubMed

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

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. PMID:27431614

  19. Antiproliferative and pro-apoptotic effects of Uncaria tomentosa in human medullary thyroid carcinoma cells.

    PubMed

    Rinner, Beate; Li, Zeng Xia; Haas, Helga; Siegl, Veronika; Sturm, Sonja; Stuppner, Hermann; Pfragner, Roswitha

    2009-11-01

    Medullary thyroid carcinoma (MTC), a rare calcitonin-producing tumor, is derived from parafollicular C-cells of the thyroid and is characterized by constitutive Bcl-2 overexpression. The tumor is relatively insensitive to radiation therapy as well as conventional chemotherapy. To date, the only curative treatment is the early and complete surgical removal of all neoplastic tissue. In this study, the antiproliferative and pro-apoptotic effects of fractions obtained from Uncaria tomentosa (Willd.) DC, commonly known as uña de gato or cat's claw were investigated. Cell growth of MTC cells as well as enzymatic activity of mitochondrial dehydrogenase was markedly inhibited after treatment with different fractions of the plant. Furthermore, there was an increase in the expressions of caspase-3 and -7 and poly(ADP-ribose) polymerase (PARP) fraction, while bcl-2 overexpression remained constant. In particular, the alkaloids isopterpodine and pteropodine of U. tomentosa exhibited a significant pro-apoptotic effect on MTC cells, whereas the alkaloid-poor fraction inhibited cell proliferation but did not show any pro-apoptotic effects. These promising results indicate the growth-restraining and apoptotic potential of plant extracts against neuroendocrine tumors, which may add to existing therapies for cancer. PMID:20032400

  20. Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

    PubMed Central

    Rieger, Daniela; Assinger, Alice; Einfinger, Katrin; Sokolikova, Barbora; Geiger, Margarethe

    2014-01-01

    Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal. PMID:25000564

  1. Cytoplasmic myosin exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability

    PubMed Central

    Cui, Xiaoxuan; Zhang, Lu; Magli, Amanda R.; Catera, Rosa; Yan, Xiao-Jie; Griffin, Daniel O.; Rothstein, Thomas L.; Barrientos, Jacqueline; Kolitz, Jonathan E.; Allen, Steven L.; Rai, Kanti R.; Chiorazzi, Nicholas; Chu, Charles C.

    2015-01-01

    The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease. PMID:26220042

  2. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

    PubMed Central

    Millet, Arnaud; Martin, Katherine R.; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A.; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

    2015-01-01

    Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

  3. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis.

    PubMed

    Millet, Arnaud; Martin, Katherine R; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

    2015-11-01

    Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

  4. Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors

    PubMed Central

    Johnson, Sara; Chen, Hexin; Lo, Pang-Kuo

    2016-01-01

    The isolation of cancer epithelial cells from mouse mammary tumor is accomplished by digestion of the solid tumor. Red blood cells and other contaminates are removed using several washing techniques such that primary epithelial cells can further enriched. This procedure yields primary tumor cells that can be used for in vitro tissue culture, fluorescence-activated cell sorting (FACS) and a wide variety of other experiments (Lo et al., 2012).

  5. Cadmium Induces p53-Dependent Apoptosis in Human Prostate Epithelial Cells

    PubMed Central

    Aimola, Pierpaolo; Carmignani, Marco; Volpe, Anna Rita; Di Benedetto, Altomare; Claudio, Luigi; Waalkes, Michael P.; van Bokhoven, Adrie; Tokar, Erik J.; Claudio, Pier Paolo

    2012-01-01

    Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl2 and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl2 concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis. PMID:22448262

  6. Effects of PACAP on intracellular signaling pathways in human retinal pigment epithelial cells exposed to oxidative stress.

    PubMed

    Fabian, E; Reglodi, D; Mester, L; Szabo, A; Szabadfi, K; Tamas, A; Toth, G; Kovacs, K

    2012-11-01

    The integrity of retinal pigment epithelial cells is critical for photoreceptor survival and vision. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts retinoprotective effects against several types of injuries in vivo, including optic nerve transection, retinal ischemia, excitotoxic injuries, UVA-induced lesion, and diabetic retinopathy. In a recent study, we have proven that PACAP is also protective in oxidative stress-induced injury in human pigment epithelial cells (ARPE-19 cells). The aim of the present study was to investigate the possible mechanisms of this protection. ARPE cells were exposed to a 24-h hydrogen peroxide treatment. Expressions of kinases and apoptotic markers were studied by complex array kits and Western blot. Oxidative stress induced the activation of several apoptotic markers, including Bad, Bax, HIF-1α, several heat shock proteins, TNF-related apoptosis-inducing ligand, and Fas-associated protein with death domain, while PACAP treatment decreased them. The changes in the expression of MAP kinases showed that PACAP activated the protective ERK1/2 and downstream CREB, and decreased the activation of the pro-apoptotic p38MAPK and c-Jun N-terminal kinase, an effect opposite to that observed with only oxidative stress. Furthermore, PACAP increased the activation of the protective Akt pathway. In addition, the effects of oxidative stress on several other signaling molecules were counteracted by PACAP treatment (Chk2, Yes, Lyn, paxillin, p53, PLC, STAT4, RSK). These play a role in cell death, cell cycle, inflammation, adhesion, differentiation and proliferation. In summary, PACAP, acting at several levels, influences the balance between pro- and anti-apoptotic factors in favor of anti-apoptosis, thereby providing protection in oxidative stress-induced injury of human retinal pigment epithelial cells. PMID:22644900

  7. The apoptotic effects of escin in the H-Ras transformed 5RP7 cell line.

    PubMed

    Güney, G; Kutlu, H M; Işcan, A

    2013-06-01

    Extracts of Aesculus hippocastanum L. (horse chestnut) seed have been used in the treatment of chronic venous insufficiency, edema and hemorrhoids. Most of the beneficial effects of horse chestnut are attributed to its principal component β-escin or escin. We have evaluated the cytotoxic and apoptotic effects of escin in the H-Ras 5RP7 cell line by analyzing cell growth inhibition, apoptosis and caspase-3 dependent activity. We have also shown structural and ultrastructural changes in these cell using confocal and transmission electron microscopy. The results indicated that escin has significant inhibitory effects on cell growth and the percentage of apoptotic cells increased after treatment with escin, and the micrographs confirmed that escin damaged these cells and induced apoptosis. PMID:22911540

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  10. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

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

    2016-01-01

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

  11. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

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

    2016-04-01

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

  12. Pro-apoptotic cell death genes, hid and reaper, from the tephritid pest species, Anastrepha suspensa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pro-apoptotic proteins from the reaper, hid, grim (RHG) family are primary regulators of programmed cell death in Drosophila due to their antagonistic effect on inhibitor of apoptosis (IAP) proteins, thereby releasing IAP inhibition of caspases that effect apoptosis. Using a degenerate PCR approach ...

  13. Inhibition of citrinin-induced apoptotic biochemical signaling in human hepatoma G2 cells by resveratrol.

    PubMed

    Chen, Chia-Chi; Chan, Wen-Hsiung

    2009-10-01

    The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis, but its precise regulatory mechanisms of action are currently unclear. Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties. In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN. Resveratrol inhibited CTN-induced ROS generation, activation of JNK, loss of mitochondrial membrane potential (MMP), as well as activation of caspase-9, caspase-3 and PAK2. Moreover, resveratrol and the ROS scavengers, NAC and alpha-tocopherol, abolished CTN-stimulated intracellular oxidative stress and apoptosis. Active JNK was required for CTN-induced mitochondria-dependent apoptotic biochemical changes, including loss of MMP, and activation of caspases and PAK2. Activation of PAK2 was essential for apoptosis triggered by CTN. These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects. PMID:20111678

  14. Artocarpus communis Induces Autophagic Instead of Apoptotic Cell Death in Human Hepatocellular Carcinoma Cells.

    PubMed

    Tzeng, Cheng-Wei; Tzeng, Wen-Sheng; Lin, Liang-Tzung; Lee, Chiang-Wen; Yen, Ming-Hong; Yen, Feng-Lin; Lin, Chun-Ching

    2015-01-01

    For centuries, natural plant extracts have played an important role in traditional medicine for curing and preventing diseases. Studies have revealed that Artocarpus communis possess various bioactivities, such as anti-inflammation, anti-oxidant, and anticancer activities. A. communis offers economic value as a source of edible fruit, yields timber, and is widely used in folk medicines. However, little is known about its molecular mechanisms of anticancer activity. Here, we demonstrate the antiproliferative activity of A. communis methanol extract (AM) and its dichloromethane fraction (AD) in two human hepatocellular carcinoma (HCC) cell lines, HepG2 and PLC/PRF/5. Colony assay showed the long-term inhibitory effect of both extracts on cell growth. DNA laddering and immunoblotting analyses revealed that both extracts did not induce apoptosis in the hepatoma cell lines. AM and AD-treated cells demonstrated different cell cycle distribution compared to UV-treated cells, which presented apoptotic cell death with high sub-G1 ratio. Instead, acridine orange staining revealed that AM and AD triggered autophagosome accumulation. Immunoblotting showed a significant expression of autophagy-related proteins, which indicated the autophagic cell death (ACD) of hepatoma cell lines. This study therefore demonstrates that A. communis AM and its dichloromethane fraction can induce ACD in HCC cells and elucidates the potential of A. communis extracts for development as anti tumor therapeutic agents that utilize autophagy as mechanism in mediating cancer cell death. PMID:25967668

  15. Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network

    PubMed Central

    Ahmed, Shafiq Uddin; Milner, Jo

    2009-01-01

    Background The regulation of apoptosis under basal (non-stress) conditions is crucial for normal mammalian development and also for normal cellular turnover in different tissues throughout life. Deficient regulation of basal apoptosis, or its perturbation, can result in impaired development and/or disease states including cancer. In contrast to stress-induced apoptosis the regulation of apoptosis under basal conditions is poorly understood. To address this issue we have compared basal- and stress-induced apoptosis in human epithelial cells of normal and cancerous origins. For this purpose we focussed our study on the opposing pro-apoptotic JNK/anti-apoptotic NFκB pathways. Methodology/Principal Findings Combinatorial RNAi plus gene knockout were employed to access and map basal regulatory pathways of apoptosis. Follow-on, in-depth analyses included exogenous expression of phosphorylation mutants and chromatin immunoprecipitation. We demonstrate that basal apoptosis is constitutively suppressed by JNK2 in a range of human cancer cell lines. This effect was not observed in non-cancer cells. Silencing JNK2 by RNAi resulted in JNK1-dependent apoptosis of cancer cells via up-regulation of the AP-1 factor c-Jun. Unexpectedly we discovered that JNK1 and c-Jun promote basal apoptosis in the absence of “activating phosphorylations” typically induced by stress. Hypo-phosphorylated c-Jun accumulated to high levels following JNK2 silencing, auto-regulated its own expression and suppressed expression of Bcl-3, an unusual IκB protein and regulator of NFκB. Basal apoptosis was mediated by components of the TNFα response pathway but was mechanistically distinct from TNFα-induced apoptosis. Conclusions/Significance Our results demonstrate that mechanistically distinct pathways operate to regulate apoptosis in mammalian cells under basal (physiological) versus stress-induced conditions. We also describe a novel apoptotic network which governs the basal survival of cancer

  16. An African swine fever virus Bc1-2 homolog, 5-HL, suppresses apoptotic cell death.

    PubMed Central

    Afonso, C L; Neilan, J G; Kutish, G F; Rock, D L

    1996-01-01

    Here, we show that the African swine fever virus 5-HL gene is a highly conserved viral gene and contains all known protein domains associated with Bcl-2 activity, including those involved with dimerization, mediating cell death, and protein-binding functions, and that its protein product, p21, suppresses apoptotic cell death in the mammalian lymphoid cell line FL5.12. Thus, 5-HL is a true functional viral member of the Bcl-2 gene family. PMID:8676523

  17. An African swine fever virus Bc1-2 homolog, 5-HL, suppresses apoptotic cell death.

    PubMed

    Afonso, C L; Neilan, J G; Kutish, G F; Rock, D L

    1996-07-01

    Here, we show that the African swine fever virus 5-HL gene is a highly conserved viral gene and contains all known protein domains associated with Bcl-2 activity, including those involved with dimerization, mediating cell death, and protein-binding functions, and that its protein product, p21, suppresses apoptotic cell death in the mammalian lymphoid cell line FL5.12. Thus, 5-HL is a true functional viral member of the Bcl-2 gene family. PMID:8676523

  18. Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis

    PubMed Central

    Ewald, Andrew J.; Brenot, Audrey; Duong, Myhanh; Chan, Bianca S.; Werb, Zena

    2009-01-01

    Summary Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly during morphogenesis, suggesting common mechanisms of epithelial growth. PMID:18410732

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

    PubMed Central

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

    2015-01-01

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

  20. Effect of blue light emitting diodes on melanoma cells: involvement of apoptotic signaling.

    PubMed

    Oh, Phil-Sun; Na, Kyung Suk; Hwang, Hyosook; Jeong, Hwan-Seok; Lim, SeokTae; Sohn, Myung-Hee; Jeong, Hwan-Jeong

    2015-01-01

    The present study was undertaken to examine whether blue LED irradiation induces cellular apoptosis in B16-F10 cells and whether it blocks the early growth of melanoma cells in mice. Irradiation with blue LED was observed to reduce cell viability and to induce apoptotic cell death, as accompanied by exposure of phosphatidylserine on the plasma outside membrane and an accumulation of a sub-G1 population. Furthermore, the mitochondrial membrane potential increased, and mitochondria-related apoptotic proteins (cytochrome c, caspase 3, and PARP) were observed. In addition, the level of intracellular superoxide anion (O2(-)) gradually increased. Interestingly the phosphorylation of p53 increased at earlier times under blue LED irradiation, but reduced after exposure for a longer time. Additionally, the thickness of the mice footpad injected with B16-F10 cells decreased significantly until the 9th day of blue LED irradiation, indicating the inhibition of the early growth rate of the melanoma cells. Our data demonstrate that blue LED irradiation induces apoptotic cell death by activating the mitochondria-mediated pathway and reduces the early growth rate of melanoma cells. Further studies are needed to elucidate the precise mechanism of blue LED in melanoma cells. PMID:25550119

  1. Apoptotic regulation and mutagenesis in human cells exposes to charged particles of importance for spaceflight

    NASA Astrophysics Data System (ADS)

    Kronenberg, A.; Gauny, S.; Hain, J.; Wu, P.; Wiese, C.

    Exposure to ionizing radiation can elicit two modes of cell death - necrosis or apoptosis. In human lymphoid cells, the predominant mechanism of radiation- induced cell death is apoptosis. The most likely exposure of individual human cells to heavy ions (e.g. Fe or Si) during spaceflight will result from single particle traversals. Here we report the fluence-response for apoptosis in human TK6 B- lymp hoblasts and provide evidence that single Fe ion traversals can stimulate an apoptotic response. The apoptotic response to charged particle exposures includes scrambling of the phospholipid bilayer in the cell membrane, activation of caspase signaling cascades and degradation of DNA into oligonucleosomes. We have also explored the importance of apoptotic regulation on the frequency and spectrum of mutations arising after exposure to charged particles. We used isogenic derivatives of TK6 cells stably transfected with pSFFV-neo-bcl-xL (encoding the anti-apoptotic gene BCL-XL and the neomycin resistance gene) or with pSFFV neo (encoding only- the neomycin resistance gene). TK6-bclxL cells were more susceptible to mutations at the TK1 locus than TK6-neo cells following exposure to protons, silicon ions or Fe ions. Molecular analysis demonstrated that most Fe-ion-induced mutations arose by loss of heterozygosity (LOH). In TK6-bclxL cells, more of the LOH occurred via mitotic recombination than in TK6-neo cells where the predominant mode of LOH was via deletion. We are currently mapping the LOH tracts to further define the biological bases for the differential sensitivity to Fe-ion-induced mutagenesis as a function of the genotype of the cell at risk. Supported by NASA grant T-964W to A. Kronenberg

  2. Selective apoptotic cell death effects of oral cancer cells treated with destruxin B

    PubMed Central

    2014-01-01

    Background Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent. Methods Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses. Results The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. Conclusions This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications. PMID:24972848

  3. Loss of α(E)-catenin promotes Fas mediated apoptosis in tubular epithelial cells

    PubMed Central

    Wang, Xinhui; Parrish, Alan R.

    2015-01-01

    The aging kidney undergoes structural and functional alterations which make it more susceptible to drug-induced acute kidney injury (AKI). Previous studies in our lab have shown that the expression of α(E)-catenin is decreased in aged kidney and loss of α(E)-catenin potentiates AKI-induced apoptosis, but not necrosis, in renal tubular epithelial cells (NRK-52E cells). However, the specific apoptotic pathway underlying the increased AKI-induced cell death is not yet understood. In this study, cells were challenged with nephrotoxicant cisplatin to induce AKI. A ~5.5-fold increase in Fas expression in C2 (stable α(E)-catenin knockdown) relative to NT3 (non-targeted control) cells was seen. Increased caspase-8 and -9 activation was induced by cisplatin in C2 as compared to NT3 cells. In addition, decreased Bcl-2 expression and increased BID cleavage and cytochrome C release were detected in C2 cells after cisplatin challenge. Treating the cells with cisplatin, in combination with a Bcl-2 inhibitor, decreased the viability of NT3 cells to the same level as C2 cells after cisplatin. Furthermore, caspase-3/-7 activation is blocked by Fas, caspase-8, caspase-9 and pan-caspase inhibitors. These inhibitors also completely abolished the difference in viability between NT3 and C2 cells in response to cisplatin. These results demonstrate a Fas-mediated apoptotic signaling pathway that is enhanced by the age-dependent loss of α(E)-catenin in renal tubule epithelial cells. PMID:25894537

  4. Caspase levels and execution efficiencies determine the apoptotic potential of the cell.

    PubMed

    Florentin, Anat; Arama, Eli

    2012-02-20

    Essentially, all metazoan cells can undergo apoptosis, but some cells are more sensitive than others to apoptotic stimuli. To date, it is unclear what determines the apoptotic potential of the cell. We set up an in vivo system for monitoring and comparing the activity levels of the two main effector caspases in Drosophila melanogaster, Drice and Dcp-1. Both caspases were activated by the apoptosome after irradiation. However, whereas each caspase alone could induce apoptosis, Drice was a more effective inducer of apoptosis than Dcp-1, which instead had a role in establishing the rate of cell death. These functional differences are attributed to their intrinsic properties rather than merely their tissue specificities. Significantly, the levels of the procaspases are directly proportional to their activity levels and play a key role in determining the cell's sensitivity to apoptosis. Finally, we provide evidence for the existence of a cellular execution threshold of caspase activity, which must be reached to induce apoptosis. PMID:22351928

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

    PubMed

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

    2015-11-01

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

  6. The suppressor of cytokine signaling SOCS1 promotes apoptosis of intestinal epithelial cells via p53 signaling in Crohn's disease.

    PubMed

    Cui, Xiaopeng; Shan, Xiaohang; Qian, Ji; Ji, Qianqian; Wang, Liang; Wang, Xiaotong; Li, Manhua; Ding, Haifang; Liu, Qingqing; Chen, Lingling; Zhang, Dongmei; Ni, Runzhou

    2016-08-01

    The suppressor of cytokine signaling SOCS1 is a member of the cytokine signaling pathway inhibitor family, which is induced by the IFN-γ induced JAK signaling pathway. The expression of SOCS1 has been found to increase in Crohn's disease (CD) patients, but the role of SOCS1 in intestinal epithelium is unclear. This study was designed to investigate whether SOCS1 has a role in the death of intestinal epithelial cells and intestinal injury. The results showed that the expression of SOCS1 increased in CD patients, and the expression of SOCS1, p-p53 and PUMA increased in the mouse TNBS induced colitis model. Using IFN-γ treated HT-29 cells as an apoptotic model of intestinal epithelial cells in vitro, we confirmed that SOCS1 promoted apoptosis of intestinal epithelial cells by activating p53. In HT-29 cells which were treated with IFN-γ, the interaction between p53 and SOCS1 and phosphorylation of p53 were significantly higher than untreated cells. When knocking SOCS1 down by using SOCS1 siRNA, phosphorylation of p53 and apoptosis of intestinal epithelial cells which was induced by IFN-γ were significantly inhibited. In summary, our findings suggest that SOCS1 may promote apoptosis of intestinal epithelial cells at least partly through mediating p53 signaling. PMID:27236107

  7. SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

    PubMed Central

    Ahmad, Shama; Nichols, David P.; Strand, Matthew; Rancourt, Raymond C.; Randell, Scott H.; White, Carl W.; Ahmad, Aftab

    2011-01-01

    Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target. PMID:22096575

  8. Secretomes of apoptotic mononuclear cells ameliorate neurological damage in rats with focal ischemia

    PubMed Central

    Altmann, Patrick; Mildner, Michael; Haider, Thomas; Traxler, Denise; Beer, Lucian; Ristl, Robin; Golabi, Bahar; Gabriel, Christian; Leutmezer, Fritz; Ankersmit, Hendrik Jan

    2014-01-01

    The pursuit of targeting multiple pathways in the ischemic cascade of cerebral stroke is a promising treatment option. We examined the regenerative potential of conditioned medium derived from rat and human apoptotic mononuclear cells (MNC), rMNC apo sec and hMNC apo sec, in experimental stroke. We performed middle cerebral artery occlusion on Wistar rats and administered apoptotic MNC-secretomes intraperitoneally in two experimental settings. Ischemic lesion volumes were determined 48 hours after cerebral ischemia. Neurological evaluations were performed after 6, 24 and 48 hours. Immunoblots were conducted to analyze neuroprotective signal-transduction in human primary glia cells and neurons. Neuronal sprouting assays were performed and neurotrophic factors in both hMNC apo sec and rat plasma were quantified using ELISA. Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively. Neurological examinations revealed improvement after stroke in both treatment groups. Co-incubation of human astrocytes, Schwann cells and neurons with hMNC apo sec resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. Analysis of neurotrophic factors in hMNC apo sec and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF). Our data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke. PMID:25383184

  9. p53 Dependent Apoptotic Cell Death Induces Embryonic Malformation in Carassius auratus under Chronic Hypoxia

    PubMed Central

    Dasgupta, Subrata; Sawant, Bhawesh T.; Chadha, Narinder K.; Pal, Asim K.

    2014-01-01

    Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos. PMID:25068954

  10. Coupling of the cell cycle and apoptotic machineries in developing T cells.

    PubMed

    Xue, Ling; Sun, Yuefang; Chiang, Leslie; He, Bo; Kang, Chulho; Nolla, Hector; Winoto, Astar

    2010-03-01

    Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4(+)CD8(+) thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4(+)CD8(+) thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4(+)CD8(+) thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4(+)CD8(+) thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis. PMID:20068041

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

    PubMed

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

    2015-10-01

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

  12. Staphylococcal Esx Proteins Modulate Apoptosis and Release of Intracellular Staphylococcus aureus during Infection in Epithelial Cells

    PubMed Central

    Korea, Charalampia G.; Balsamo, Giuliana; Pezzicoli, Alfredo; Merakou, Christina; Tavarini, Simona; Bagnoli, Fabio; Serruto, Davide

    2014-01-01

    The opportunistic pathogen Staphylococcus aureus is one of the major causes of health care-associated infections. S. aureus is primarily an extracellular pathogen, but it was recently reported to invade and replicate in several host cell types. The ability of S. aureus to persist within cells has been implicated in resistance to antimicrobials and recurrent infections. However, few staphylococcal proteins that mediate intracellular survival have been identified. Here we examine if EsxA and EsxB, substrates of the ESAT-6-like secretion system (Ess), are important during intracellular S. aureus infection. The Esx proteins are required for staphylococcal virulence, but their functions during infection are unclear. While isogenic S. aureus esxA and esxB mutants were not defective for epithelial cell invasion in vitro, a significant increase in early/late apoptosis was observed in esxA mutant-infected cells compared to wild-type-infected cells. Impeding secretion of EsxA by deleting C-terminal residues of the protein also resulted in a significant increase of epithelial cell apoptosis. Furthermore, cells transfected with esxA showed an increased protection from apoptotic cell death. A double mutant lacking both EsxA and EsxB also induced increased apoptosis but, remarkably, was unable to escape from cells as efficiently as the single mutants or the wild type. Thus, using in vitro models of intracellular staphylococcal infection, we demonstrate that EsxA interferes with host cell apoptotic pathways and, together with EsxB, mediates the release of S. aureus from the host cell. PMID:25047846

  13. Cell Chirality Induces Collective Cell Migration in Epithelial Sheets

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Shibata, Tatsuo

    2015-10-01

    During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

  14. Fungal glycan interactions with epithelial cells in allergic airway disease

    PubMed Central

    Roy, René M.; Klein, Bruce S.

    2014-01-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. PMID:23602359

  15. Methionine deficiency reduces autophagy and accelerates death in intestinal epithelial cells infected with enterotoxigenic Escherichia coli.

    PubMed

    Tang, Yulong; Tan, Bie; Xiong, Xia; Li, Fengna; Ren, Wenkai; Kong, Xiangfeng; Qiu, Wei; Hardwidge, Philip R; Yin, Yulong

    2015-10-01

    Infections by enterotoxigenic Escherichia coli (ETEC) result in large economic losses to the swine industry worldwide. Dietary supplementation with amino acids has been considered as a potential mechanism to improve host defenses against infection. The goal of this study was to determine whether methionine deprivation alters ETEC interactions with porcine intestinal epithelial cells. IPEC-1 cells were cultured in media with or without L-methionine. Methionine deprivation resulted in enhanced ETEC adhesion and increased both the cytotoxicity and apoptotic responses of IPEC-1 cells infected with ETEC. Methionine deprivation inhibited IPEC-1 cell autophagic responses, suggesting that the increased cytotoxicity of ETEC to methionine-deprived IPEC-1 cells might be due to defects in autophagy. PMID:24965529

  16. Quantitative Assessment of Cytosolic Salmonella in Epithelial Cells

    PubMed Central

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

    2014-01-01

    Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol. PMID:24400108

  17. Lung epithelial cells modulate the inflammatory response of alveolar macrophages.

    PubMed

    Rubovitch, Vardit; Gershnabel, Shoham; Kalina, Moshe

    2007-12-01

    The goal of this study was to examine the effect of alveolar epithelial cells on inflammatory responses in macrophages. Lung epithelial cells (either rat RLE-6TN or human A549 cells) reduced LPS-induced NO production in alveolar macrophages (AM) in a contact-independent mechanism. The inhibitory effect of the epithelial cells was present already at the transcriptional level: LPS-induced inducible NO synthase (iNOS) expression was significantly smaller. Surfactant protein A (SP-A)-induced NO production by alveolar macrophages was also reduced in the presence of A549 cells, though, by a different kinetics. LPS-induced interleukin-6 (IL-6) production (another inflammatory pathway) by alveolar macrophages was also reduced in the presence of RLE-6TN cells. These data suggest a role for lung epithelial cells in the complicated modulation of inflammatory processes, and provide an insight into the mechanism underlying. PMID:17851743

  18. Detection of Apoptotic Versus Autophagic Cell Death by Flow Cytometry.

    PubMed

    Sica, Valentina; Maiuri, M Chiara; Kroemer, Guido; Galluzzi, Lorenzo

    2016-01-01

    Different modes of regulated cell death (RCD) can be initiated by distinct molecular machineries and their morphological manifestations can be difficult to discriminate. Moreover, cells responding to stress often activate an adaptive response centered around autophagy, and whether such a response is cytoprotective or cytotoxic cannot be predicted based on morphological parameters only. Molecular definitions are therefore important to understand various RCD subroutines from a mechanistic perspective. In vitro, various forms of RCD including apoptosis and autophagic cell death can be easily discriminated from each other with assays that involve chemical or pharmacological interventions targeting key components of either pathway. Here, we detail a straightforward method to discriminate apoptosis from autophagic cell death by flow cytometry, based on the broad-spectrum caspase inhibitor Z-VAD-fmk and the genetic inhibition of ATG5. PMID:27108427

  19. Dietary antioxidants protect gut epithelial cells from oxidant-induced apoptosis

    PubMed Central

    Miller, Mark JS; Angeles, Fausto M; Reuter, Brian K; Bobrowski, Paul; Sandoval, Manuel

    2001-01-01

    Background The potential of ascorbic acid and two botanical decoctions, green tea and cat's claw, to limit cell death in response to oxidants were evaluated in vitro. Methods Cultured human gastric epithelial cells (AGS) or murine small intestinal epithelial cells (IEC-18) were exposed to oxidants – DPPH (3 μM), H2O2 (50 μM), peroxynitrite (300 μM) – followed by incubation for 24 hours, with antioxidants (10 μg/ml) administered as a 1 hour pretreatment. Cell number (MTT assay) and death via apoptosis or necrosis (ELISA, LDH release) was determined. The direct interactions between antioxidants and DPPH (100 μM) or H2O2 (50 μM) were evaluated by spectroscopy. Results The decoctions did not interact with H2O2, but quenched DPPH although less effectively than vitamin C. In contrast, vitamin C was significantly less effective in protecting human gastric epithelial cells (AGS) from apoptosis induced by DPPH, peroxynitrite and H2O2 (P < 0.001). Green tea and cat's claw were equally protective against peroxynitrite and H2O2, but green tea was more effective than cat's claw in reducing DPPH-induced apoptosis (P < 0.01). Necrotic cell death was marginally evident at these low concentrations of peroxynitrite and H2O2, and was attenuated both by cat's claw and green tea (P < 0.01). In IEC-18 cells, all antioxidants were equally effective as anti-apoptotic agents. Conclusions These results indicate that dietary antioxidants can limit epithelial cell death in response to oxidant stress. In the case of green tea and cat's claw, the cytoprotective response exceed their inherent ability to interact with the injurious oxidant, suggestive of actions on intracellular pathways regulating cell death. PMID:11749672

  20. Ferroptosis: An Iron-Dependent Form of Non-Apoptotic Cell Death

    PubMed Central

    Dixon, Scott J.; Lemberg, Kathryn M.; Lamprecht, Michael R.; Skouta, Rachid; Zaitsev, Eleina M.; Gleason, Caroline E.; Patel, Darpan N.; Bauer, Andras J.; Cantley, Alexandra M.; Yang, Wan Seok; Morrison, Barclay; Stockwell, Brent R.

    2012-01-01

    SUMMARY Non-apoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of non-apoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically and genetically distinct from apoptosis, necrosis and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system xc−), creating a void in the antioxidant defenses of the cell, ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the non-apoptotic destruction of certain cancer cells, while inhibition of this process may protect organisms from neurodegeneration. PMID:22632970

  1. PDR-1/hParkin negatively regulates the phagocytosis of apoptotic cell corpses in Caenorhabditis elegans

    PubMed Central

    Cabello, J; Sämann, J; Gómez-Orte, E; Erazo, T; Coppa, A; Pujol, A; Büssing, I; Schulze, B; Lizcano, J M; Ferrer, I; Baumeister, R; Dalfo, E

    2014-01-01

    Apoptotic cell death is an integral part of cell turnover in many tissues, and proper corpse clearance is vital to maintaining tissue homeostasis in all multicellular organisms. Even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. In Caenorhabditis elegans, two parallel and partly redundant conserved pathways act in cell corpse engulfment. The pathway for cytoskeletal rearrangement requires the small GTPase CED-10 Rac1 acting for an efficient surround of the dead cell. The CED-10 Rac pathway is also required for the proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. Parkin, the mammalian homolog of the C. elegans PDR-1, interacts with Rac1 in aged human brain and it is also implicated with actin dynamics and cytoskeletal rearrangements in Parkinsons's disease, suggesting that it might act on engulfment. Our genetic and biochemical studies indicate that PDR-1 inhibits apoptotic cell engulfment and DTC migration by ubiquitylating CED-10 for degradation. PMID:24625979

  2. Akebia saponin PA induces autophagic and apoptotic cell death in AGS human gastric cancer cells.

    PubMed

    Xu, Mei-Ying; Lee, Dong Hwa; Joo, Eun Ji; Son, Kun Ho; Kim, Yeong Shik

    2013-09-01

    In this study, we investigated the anticancer mechanism of akebia saponin PA (AS), a natural product isolated from Dipsacus asperoides in human gastric cancer cell lines. It was shown that AS-induced cell death is caused by autophagy and apoptosis in AGS cells. The apoptosis-inducing effect of AS was characterized by annexin V/propidium (PI) staining, increase of sub-G1 phase and caspase-3 activation, while the autophagy-inducing effect was indicated by the formation of cytoplasmic vacuoles and microtubule-associated protein 1 light chain-3 II (LC3-II) conversion. The autophagy inhibitor bafilomycin A1 (BaF1) decreased AS-induced cell death and caspase-3 activation, but caspase-3 inhibitor Ac-DEVD-CHO did not affect LC3-II accumulation or AS-induced cell viability, suggesting that AS induces autophagic cell death and autophagy contributes to caspase-3-dependent apoptosis. Furthermore, AS activated p38/c-Jun N-terminal kinase (JNK), which could be inhibited by BaF1, and caspase-3 activation was attenuated by both SB202190 and SP600125, indicating that AS-induced autophagy promotes mitogen-activated protein kinases (MAPKs)-mediated apoptosis. Taken together, these results demonstrate that AS induces autophagic and apoptotic cell death and autophagy plays the main role in akebia saponin PA-induced cell death. PMID:23850994

  3. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

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

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

  4. Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

    PubMed

    Alaimo, Agustina; Gorojod, Roxana M; Miglietta, Esteban A; Villarreal, Alejandro; Ramos, Alberto J; Kotler, Mónica L

    2013-10-25

    Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment. PMID:24021799

  5. Intrinsic epithelial cells repair the kidney after injury.

    PubMed

    Humphreys, Benjamin D; Valerius, M Todd; Kobayashi, Akio; Mugford, Joshua W; Soeung, Savuth; Duffield, Jeremy S; McMahon, Andrew P; Bonventre, Joseph V

    2008-03-01

    Understanding the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease. The kidney can repair after even a severe insult, but whether adult stem or progenitor cells contribute to epithelial renewal after injury and the cellular origin of regenerating cells remain controversial. Using genetic fate-mapping techniques, we generated transgenic mice in which 94%-95% of tubular epithelial cells, but no interstitial cells, were labeled with either beta-galactosidase (lacZ) or red fluorescent protein (RFP). Two days after ischemia-reperfusion injury (IRI), 50.5% of outer medullary epithelial cells coexpress Ki67 and RFP, indicating that differentiated epithelial cells that survived injury undergo proliferative expansion. After repair was complete, 66.9% of epithelial cells had incorporated BrdU, compared to only 3.5% of cells in the uninjured kidney. Despite this extensive cell proliferation, no dilution of either cell-fate marker was observed after repair. These results indicate that regeneration by surviving tubular epithelial cells is the predominant mechanism of repair after ischemic tubular injury in the adult mammalian kidney. PMID:18371453

  6. Clinical implications of epithelial cell plasticity in cancer progression.

    PubMed

    Aparicio, Luis A; Blanco, Moisés; Castosa, Raquel; Concha, Ángel; Valladares, Manuel; Calvo, Lourdes; Figueroa, Angélica

    2015-09-28

    In the last few years, the role of epithelial cell plasticity in cancer biology research has gained increasing attention. This concept refers to the ability of the epithelial cells to dynamically switch between different phenotypic cellular states. This programme is particularly relevant during the epithelial-to-mesenchymal transition (EMT) in cancer progression. During colonization, epithelial cells first activate the EMT programme to disseminate from a primary tumour to reach a distant tissue site. During this process, cells are transported into the circulation and are able to escape the immune system of the host. Then, a reverse process called mesenchymal-to-epithelial transition (MET) occurs on cells that settle in the distant organs. Although epithelial cell plasticity has an important impact on tumour biology, the clinical relevance of this concept remains to be recapitulated. In this review, we will update the current state of epithelial cell plasticity in cancer progression and its clinical implications for the design of therapeutic strategies, the acquisition of multidrug resistance, and future perspectives for the management of cancer patients. PMID:26099173

  7. Liver epithelial cells inhibit proliferation and invasiveness of hepatoma cells.

    PubMed

    Jeng, Kuo-Shyang; Jeng, Chi-Juei; Jeng, Wen-Juei; Sheen, I-Shyan; Li, Shih-Yun; Hung, Zih-Hang; Hsiau, Hsin-I; Yu, Ming-Che; Chang, Chiung-Fang

    2016-03-01

    Hepatocellular carcinoma (HCC) is a worldwide malignancy with poor prognosis. Liver progenitors or stem cells could be a potential therapy for HCC treatment since they migrate toward tumors. Rat liver epithelial (RLE) cells have both progenitor and stem cell-like properties. Therefore, our study elucidated the therapeutic effect of RLE cells in rat hepatoma cells. RLE cells were isolated from 10-day old rats and characterized for stem cell marker expression. RLE cells and rat hepatoma cells (H4-IIE-C3 cells) were co-cultured and divided into four groups with different ratios of RLE and hepatoma cells. Group A had only rat hepatoma cells as a control group. The ratios of rat hepatoma and RLE cells in group B, C and D were 5:1, 1:1 and 1:5, respectively. Effective inhibition of cell proliferation and migration was found in group D when compared to group A. There was a significant decrease in Bcl2 expression and increase in late apoptosis of rat hepatoma cells when adding more RLE cells. RLE cells reduced cell proliferation and migration of rat hepatoma cells. These results suggested that RLE cells could be used as a potential cell therapy. PMID:26647726

  8. Sodium selectivity of Reissner's membrane epithelial cells

    PubMed Central

    2011-01-01

    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 media. PMID:21284860

  9. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

    PubMed Central

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIA-BLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

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

    PubMed

    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. PMID:25963259

  11. Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay

    PubMed Central

    Wunsch, Marie; Caspell, Richard; Kuerten, Stefanie; Lehmann, Paul V.; Sundararaman, Srividya

    2015-01-01

    As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay. PMID:25585298

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

    PubMed Central

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

    2014-01-01

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

  13. Sepsis-associated AKI: epithelial cell dysfunction.

    PubMed

    Emlet, David R; Shaw, Andrew D; Kellum, John A

    2015-01-01

    Acute kidney injury (AKI) occurs frequently in critically ill patients with sepsis, in whom it doubles the mortality rate and half of the survivors suffer permanent kidney damage or chronic kidney disease. Failure in the development of viable therapies has prompted studies to better elucidate the cellular and molecular etiologies of AKI, which have generated novel theories and paradigms for the mechanisms of this disease. These studies have shown multifaceted origins and elements of AKI that, in addition to/in lieu of ischemia, include the generation of damage-associated molecular patterns and pathogen-associated molecular patterns, the inflammatory response, humoral and cellular immune activation, perturbation of microvascular flow and oxidative stress, bioenergetic alterations, cell-cycle alterations, and cellular de-differentiation/re-differentiation. It is becoming clear that a major etiologic effector of all these inputs is the renal tubule epithelial cell (RTEC). This review discusses these elements and their effects on RTECs, and reviews the current hypotheses of how these effects may determine the fate of RTECs during sepsis-induced AKI. PMID:25795502

  14. BCL2 suppresses PARP1 function and non-apoptotic cell death

    PubMed Central

    Dutta, Chaitali; Day, Tovah; Kopp, Nadja; van Bodegom, Diederik; Davids, Matthew S.; Ryan, Jeremy; Bird, Liat; Kommajosyula, Naveen; Weigert, Oliver; Yoda, Akinori; Fung, Hua; Brown, Jennifer R.; Shapiro, Geoffrey I.; Letai, Anthony; Weinstock, David M.

    2014-01-01

    BCL2 suppresses apoptosis by binding the BH3 domain of pro-apoptotic factors and thereby regulating outer mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chronic lymphocytic leukemia, are dependent on BCL2 for survival, but become resistant to apoptosis after treatment. Here we identified a direct interaction between the anti-apoptotic protein BCL2 and the enzyme poly(ADP) ribose polymerase 1 (PARP1), which suppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B cell lymphoma cells. The BH3 mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, re-establishing PARP1 activity and DNA repair and promoting non-apoptotic cell death. This form of cell death was unaffected by resistance to single-agent ABT-737 that results from upregulation of anti-apoptotic BCL2 family members. Based on the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would kill PARP inhibitor-sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor-sensitive breast cancer and lung cancer cells by 90-100%, and these effects were reversed by ABT-737. Taken together, our findings demonstrate that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity and suppresses PARP1-dependent repair. Targeted disruption of the BCL2-PARP1 interaction therefore may represent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis. PMID:22689920

  15. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  17. Investigation of the apoptotic way induced by digallic acid in human lymphoblastoid TK6 cells

    PubMed Central

    2012-01-01

    Background The digallic acid (DGA) purified from Pistacia lentiscus. L fruits was investigated for its antiproliferative and apoptotic activities on human lymphoblastoid TK6 cells. Methods We attempt to characterize the apoptotic pathway activated by DGA. Apoptosis was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activities. Results The inhibition of lymphoblastoid cell proliferation was noted from 8.5 μg/ml of DGA. The induction of apoptosis was confirmed by DNA fragmentation and PARP cleavage. We have demonstrated that DGA induces apoptosis by activating the caspase-8 extrinsic pathway. Caspase-3 was also activated in a dose dependent manner. Conclusion In summary, DGA exhibited an apoptosis inductor effect in TK6 cells revealing thus its potential as a cancer-preventive agent. PMID:22686580

  18. Pro-apoptotic NOXA is implicated in atmospheric-pressure plasma-induced melanoma cell death

    NASA Astrophysics Data System (ADS)

    Ishaq, M.; Bazaka, K.; Ostrikov, K.

    2015-11-01

    Atmospheric-pressure plasma (APP) has been successfully used to treat several types of cancers in vivo and in vitro, with the effect being primarily attributed to the generation of reactive oxygen species (ROS). However, the mechanisms by which APP induces apoptosis in cancer cells require further elucidation. In this study, the effects of APP on the expression of 500 genes in melanoma Mel007 cancer cells were examined. Pro-apoptotic phorbol-12-myristate-13-acetate-induced protein (PMAIP1), also known as NOXA, was highly expressed as a result of APP treatment in a dose-dependent manner. Blocking of ROS using scavenger NAC or silencing of NOXA gene by RNA interference inhibited the APP-induced NOXA genes upregulation and impaired caspases 3/7 mediated apoptosis, confirming the important role plasma-generated ROS species and pro-apoptotic NOXA play in APP-induced cancer cell death.

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

    PubMed

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

    2013-01-01

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions. PMID:23425702

  20. Induction of Cell Death through Alteration of Oxidants and Antioxidants in Epithelial Cells Exposed to High Energy Protons

    NASA Technical Reports Server (NTRS)

    Ramesh, Govindarajan; Wu, Honglu

    2012-01-01

    Radiation affects several cellular and molecular processes including double strand breakage, modifications of sugar moieties and bases. In outer space, protons are the primary radiation source which poses a range of potential health risks to astronauts. On the other hand, the use of proton radiation for tumor radiation therapy is increasing as it largely spares healthy tissues while killing tumor tissues. Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. Therefore, in the present study, we irradiated rat epithelial cells (LE) with different doses of protons and investigated their effects on cell proliferation and cell death. Our data showed an inhibition of cell proliferation in proton irradiated cells with a significant dose dependent activation and repression of reactive oxygen species (ROS) and antioxidants, glutathione and superoxide dismutase respectively as compared to control cells. In addition, apoptotic related genes such as caspase-3 and -8 activities were induced in a dose dependent manner with corresponding increased levels of DNA fragmentation in proton irradiated cells than control cells. Together, our results show that proton radiation alters oxidant and antioxidant levels in the cells to activate apoptotic pathway for cell death.

  1. Induction of Cell Death Through Alteration of Oxidants and Antioxidants in Epithelial Cells Exposed to High Energy Protons

    NASA Astrophysics Data System (ADS)

    Ramesh, Govindarajan; Wu, Honglu

    2012-07-01

    Radiation affects several cellular and molecular processes including double strand breakage, modifications of sugar moieties and bases. In outer space, protons are the primary radiation source which poses a range of potential health risks to astronauts. On the other hand, the use of proton radiation for tumor radiation therapy is increasing as it largely spares healthy tissues while killing tumor tissues. Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. Therefore, in the present study, we irradiated rat epithelial cells (LE) with different doses of protons and investigated their effects on cell proliferation and cell death. Our data showed an inhibition of cell proliferation in proton irradiated cells with a significant dose dependent activation and repression of reactive oxygen species (ROS) and antioxidants, glutathione and superoxide dismutase respectively as compared to control cells. In addition, apoptotic related genes such as caspase-3 and -8 activities were induced in a dose dependent manner with corresponding increased levels of DNA fragmentation in proton irradiated cells than control cells. Together, our results show that proton radiation alters oxidant and antioxidant levels in the cells to activate apoptotic pathway for cell death.

  2. Apoptotic Susceptibility to DNA Damage of Pluripotent Stem Cells Facilitates Pharmacologic Purging of Teratoma Risk

    PubMed Central

    Smith, Alyson J.; Nelson, Natalie G.; Oommen, Saji; Hartjes, Katherine A.; Folmes, Clifford D.; Terzic, Andre

    2012-01-01

    Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells. PMID:23197662

  3. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

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

    2014-01-01

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

  4. Microfluidic approaches for epithelial cell layer culture and characterisation.

    PubMed

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

    2014-07-01

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

  5. Molecular responses of rat tracheal epithelial cells to transmembrane pressure.

    PubMed

    Ressler, B; Lee, R T; Randell, S H; Drazen, J M; Kamm, R D

    2000-06-01

    Smooth muscle constriction in asthma causes the airway to buckle into a rosette pattern, folding the epithelium into deep crevasses. The epithelial cells in these folds are pushed up against each other and thereby experience compressive stresses. To study the epithelial cell response to compressive stress, we subjected primary cultures of rat tracheal epithelial cells to constant elevated pressures on their apical surface (i.e., a transmembrane pressure) and examined changes in the expression of genes that are important for extracellular matrix production and maintenance of smooth muscle activation. Northern blot analysis of RNA extracted from cells subjected to transmembrane pressure showed induction of early growth response-1 (Egr-1), endothelin-1, and transforming growth factor-beta1 in a pressure-dependent and time-dependent manner. Increases in Egr-1 protein were detected by immunohistochemistry. Our results demonstrate that airway epithelial cells respond rapidly to compressive stresses. Potential transduction mechanisms of transmembrane pressure were also investigated. PMID:10835333

  6. Alveolar Epithelial Cells Undergo Epithelial-to-Mesenchymal Transition in Response to Endoplasmic Reticulum Stress*

    PubMed Central

    Tanjore, Harikrishna; Cheng, Dong-Sheng; Degryse, Amber L.; Zoz, Donald F.; Abdolrasulnia, Rasul; Lawson, William E.; Blackwell, Timothy S.

    2011-01-01

    Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis. PMID:21757695

  7. Necrotic Effect versus Apoptotic Nature of Camptothecin in Human Cervical Cancer Cells

    PubMed Central

    Zare-Mirakabadi, Abbas; Sarzaeem, Ali; Moradhaseli, Saeed; Sayad, Aida; Negahdary, Masoud

    2012-01-01

    Background Functional defects in mitochondria are involved in the induction of cell death in cancer cells. The process of programmed cell death may occur through the mechanisms of apoptosis. Several potential lead molecules such as Camptothecin (CPT) and its analogues have been isolated from plants with anticancer effect. The aim of the present study was to understand the necrotic effect versus apoptotic nature of CPT in HeLa cancer cells. Methods The anti-proliferative activity of CPT was estimated through 3-(4, 5- Dimethyl Thiazol-2-yl)-2, 5-diphenyl Tetrazolium bromide (MTT) assay and DNA fragmentation analysis using gel electrophoresis. Lactate Dehydrogenase (LDH) activity and cell morphology were assessed under control and CPT exposed conditions to evaluate the necrotic effect of CPT. Results The results showed that CPT inhibited the proliferation of HeLa cells in a dose-dependent manner with an Inhibitory Concentration 50% (IC50) of 0.08±0.012 µg/ml. However the significant (p<0.05) increase happens in LDH activity at concentrations 1×10-1µg/ml and above. Morphological changes showed that CPT in low concentrations induced an apoptotic mechanism of cell death, such as cell shrinkage and characteristic rounding of dying cells, while at high concentrations showed necrosis changes. The characteristic DNA ladder formation of CPT-treated cells in agarose gel electrophoresis confirmed the results obtained by light microscopy and LDH assay. Conclusion Camptothecin as an anticancer drug may have anti-proliferative effect on HeLa cancer cells in low concentrations, through its nature of induction of apoptosis. The border line between necrotic effect and apoptotic nature of CPT in HeLa cancer cells has been found to be at concentration of 1×10-1 µg/ml. PMID:25628829

  8. Response of corneal epithelial cells to Staphylococcus aureus

    PubMed Central

    2010-01-01

    Staphylococcus aureus is a leading cause of invasive infection. It also infects wet mucosal tissues including the cornea and conjunctiva. Conflicting evidence exists on the expression of Toll-like receptors by human corneal epithelial cells. It was therefore of interest to determine how epithelial cells from this immune privileged tissue respond to S. aureus. Further, it was of interest to determine whether cytolytic toxins, with the potential to cause ion flux or potentially permit effector molecule movement across the target cell membrane, alter the response. Microarrays were used to globally assess the response of human corneal epithelial cells to S. aureus. A large increase in abundance of transcripts encoding the antimicrobial dendritic cell chemokine, CCL20, was observed. CCL20 release into the medium was detected, and this response was found to be largely TLR2 and NOD2 independent. Corneal epithelial cells also respond to S. aureus by increasing the intracellular abundance of mRNA for inflammatory mediators, transcription factors, and genes related to MAP kinase pathways, in ways similar to other cell types. The corneal epithelial cell response was surprisingly unaffected by toxin exposure. Toxin exposure did, however, induce a stress response. Although model toxigenic and non-toxigenic strains of S. aureus were employed in the present study, the results obtained were strikingly similar to those reported for stimulation of vaginal epithelial cells by clinical toxic shock toxin expressing isolates, demonstrating that the initial epithelial cellular responses to S. aureus are largely independent of strain as well as epithelial cell tissue source. PMID:21178447

  9. Flow Cytometry Analysis of Thymic Epithelial Cells and Their Subpopulations.

    PubMed

    Ohigashi, Izumi; Takahama, Yousuke

    2016-01-01

    The parenchyma of the thymus is compartmentalized into the cortex and the medulla, which are constructed by cortical thymic epithelial cells (cortical TECs, cTECs) and medullary thymic epithelial cells (mTECs), respectively. cTECs and mTECs essentially and differentially regulate the development and repertoire selection of T cells. Consequently, the biology of T cell development and selection includes the study of TECs in addition to the study of developing T cells and other hematopoietic cells including dendritic cells. In this chapter, we describe the methods for flow cytometric analysis and sorting of TECs and their subpopulations, including cTECs and mTECs. PMID:26294398

  10. Infection of human fallopian tube epithelial cells with Neisseria gonorrhoeae protects cells from tumor necrosis factor alpha-induced apoptosis.

    PubMed

    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

    2006-06-01

    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-alpha). 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-alpha 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-alpha antibodies; and (iii) the addition of exogenous TNF-alpha induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-alpha-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-alpha-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

  11. Neisseria gonorrhoeae infection protects human endocervical epithelial cells from apoptosis via expression of host antiapoptotic proteins.

    PubMed

    Follows, S A; Murlidharan, J; Massari, P; Wetzler, L M; Genco, C A

    2009-09-01

    Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells. PMID:19546192

  12. Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific low-molecular-weight form is associated with apoptosis in vitro.

    PubMed

    Browne, S J; Williams, A C; Hague, A; Butt, A J; Paraskeva, C

    1994-10-01

    APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1-3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36-96% depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC was also lost, though a 95-kDa protein distinct from that in apoptotic cells was observed. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival. PMID:7927905

  13. Diversity of epithelial stem cell types in adult lung.

    PubMed

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  14. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  15. Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos.

    PubMed

    Wood, W; Turmaine, M; Weber, R; Camp, V; Maki, R A; McKercher, S R; Martin, P

    2000-12-01

    Apoptosis is one of the key tools used by an embryo to regulate cell numbers and sculpt body shape. Although massive numbers of cells die during development, they are so rapidly phagocytosed that very few corpses are ever seen in most embryonic tissues. In this paper, we focus on the catastrophic cell death that occurs as the developing footplate is remodelled to transform webbed regions into free interdigital spaces. In the wild-type embryo, these dead cells are rapidly engulfed and cleared by macrophages. We show that in a macrophageless mouse embryo, null for the haemopoetic-lineage-specific transcription factor, PU.1, the task of phagocytosis is taken over by 'stand-in' mesenchymal neighbours in a clear example of cell redundancy. However, it takes three times as many of these mesenchymal phagocytes to complete the task and, at each stage of the clearance process - in the recognition of apoptotic debris, its engulfment and finally its digestion - they appear to be less efficient than macrophages. A molecular explanation for this may be that several of the engulfment genes expressed by macrophages, including the ABC1 transporter (believed to be part of the phagocytic machinery conserved from Caenorhabditis elegans to mouse), are not upregulated by these 'stand-in' phagocytes. PMID:11076747

  16. HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

    PubMed Central

    Tugizov, Sharof M.; Herrera, Rossana; Veluppillai, Piri; Greenspan, Deborah; Soros, Vanessa; Greene, Warner C.; Levy, Jay A.; Palefsky, Joel M.

    2010-01-01

    Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells – and not those that passed through the adult cells – remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission. PMID:21056450

  17. Assessment of an in vitro model of lung epithelial cell stress responses.

    PubMed

    Taylor, Mark; Vella, Laura; Carr, Tony

    2014-10-01

    The ability of cells to adapt and survive environmental and physiological stress relies on activation of cellular stress responses. These include anti-oxidant, inflammatory and apoptotic responses. These cellular responses are mediated by cell signalling pathways, including those controlled by the transcription factors Nrf2 (antioxidant response) and NF-?ß (inflammatory response). As part of a suite of in vitro models for the comparison of different nicotine delivery products, an in vitro lung cell stress response model has been developed. Human bronchial epithelial cells were exposed to positive controls or cigarette smoke aqueous extracts (CSEaq) from 3R4F and 1R5F reference cigarettes for 4hours. Cellular responses were then measured as oxidative, pro-inflammatory, apoptotic and necrotic endpoints. Cellular oxidative stress was characterised by measurement of the intracellular glutathione ratio, intracellular ROS production and activation of the Nrf2-controlled Anti-oxidative Response Elements (ARE). The inflammatory response of the cells was determined through quantification of secreted cytokines, IL-1a, IL-6 and IL-8. Apoptotic and necrotic responses were characterised by measurements of Caspase 3/7 activity and live-cell protease activity. Stably transfected luciferase reporter cell lines were utilised to quantify the transcriptional control of anti-oxidant and inflammatory pathways. All cell stress response endpoints were activated by exposure to positive controls or CSEaq. The observed concentration-dependent lowering of the glutathione ratio and increase in intracellular ROS generation corresponded with an increase in Nrf2 transcriptional activation of the ARE. It has been demonstrated that this model was able to distinguish between CSEaq from the two different reference cigarettes. We propose that this model may be suitably sensitive for biological comparisons of cigarettes against different nicotine delivery products. PMID:26461402

  18. Mechanisms of JP-8 jet fuel toxicity. I. Induction of apoptosis in rat lung epithelial cells.

    PubMed

    Stoica, B A; Boulares, A H; Rosenthal, D S; Iyer, S; Hamilton, I D; Smulson, M E

    2001-03-01

    JP-8 is a kerosene-based fuel widely used by the U.S. military. Various models of human occupational and animal exposure to JP-8 have demonstrated the potential for local and systemic toxicity but the mechanisms involved are unknown. The purpose of our investigation was to study the molecular mechanisms of JP-8 toxicity by using an in vitro model. JP-8 exposure in a rat lung alveolar type II epithelial cell line (RLE-6TN) induces biochemical and morphological markers of apoptotic cell death: caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, chromatin condensation, membrane blebbing, cytochrome c release from mitochondria, and genomic DNA cleavage into both oligonucleosomal (DNA ladder) and high-molecular-weight (HMW) fragments. The human histiocytic lymphoma cell line (U937) also responds to JP-8 with caspase-3 activation, cleavage of caspase substrates, including PARP, DNA-PK, and lamin B1, and degradation of genomic DNA with the production of HMW fragments. Caspase-3 activation and PARP cleavage also occur in the acute T-cell leukemia cell line (Jurkat) following treatment with JP-8. Furthermore, Jurkat cells stably transfected with a plasmid encoding the antiapoptotic protein Bcl-x(L) or pretreated with the pan-caspase inhibitor Boc-d-fmk, are relatively resistant to the cytotoxic effects of JP-8 compared to control cells. Finally, we demonstrate that PARP cleavage occurs in primary mouse thymocytes exposed to JP-8. In conclusion, our data support the hypothesis that apoptotic cell death is responsible at least partially for the cytotoxic effects of JP-8 and suggest that inhibition of the apoptotic cascade might reduce JP-8 toxicity. PMID:11222085

  19. The fungicide Mancozeb induces metacaspase-dependent apoptotic cell death in Saccharomyces cerevisiae BY4741.

    PubMed

    Scariot, F J; Jahn, L M; Maianti, J P; Delamare, A P L; Echeverrigaray, S

    2016-07-01

    Mancozeb (MZ), a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts, is one of the most widely used fungicides in agriculture. Toxicologic studies in mammals and mammalian cells indicate that this fungicide can cause neurological and cytological disorders, putatively associated with pro-oxidant and apoptotic effects. Yeast adaptation to sub-inhibitory concentrations of MZ has been correlated with oxidative response, proteins degradation, and energy metabolism, and its main effect on yeast has been attributed to its high reactivity with thiol groups in proteins. Herein, we show that acute MZ treatments on aerobic exponentially growing yeast of wild type (BY4741) and deletion mutant strains, coupled with multiplex flow cytometry analysis, conclusively demonstrated that MZ displays the typical features of pro-oxidant activity on Saccharomyces, elevating mitochondrial ROS, and causing hyper-polarization of mitochondrial membranes leading to apoptosis. A drastic reduction of cellular viability associated with the maintenance of cell membrane integrity, as well as phosphatidyl serine externalization on yeast cells exposed to MZ, also supports an apoptotic mode of action. Moreover, abrogation of the apoptotic response in yca1 deficient mutants indicates that metacaspase-1 is involved in the programmed cell death mechanism induced by MZ in yeast. PMID:27160815

  20. 4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

    PubMed Central

    Sharma, Abha; Sharma, Rajendra; Chaudhary, Pankaj; Vatsyayan, Rit; Pearce, Virginia; Jeyabal, Prince V.S.; Zimniak, Piotr; Awasthi, Sanjay; Awasthi, Yogesh C.

    2009-01-01

    4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (-/-) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs. PMID:18930016

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

    SciTech Connect

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

    2009-07-01

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

  2. Parvalbumin in cortical epithelial cells of the pigeon thymus

    PubMed Central

    ATOJI, YASURO; YAMAMOTO, YOSHIO; SUZUKI, YOSHITAKA

    2000-01-01

    We examined the distribution of parvalbumin in the pigeon thymus by light and electron microscopic immunohistochemistry. Tissues were also examined by conventional electron microscopy to determine the ultrastructure of immunoreactive cells. Parvalbumin immunoreaction was located in epithelial cells of the cortex, which formed dense mesh-like structures. Parvalbumin-positive epithelial cells were classified into 2 types. The first comprised elongated cells. In these, the nucleus was spindle-shaped, oval, or triangular, with a slightly irregular contour and contained rich heterochromatin peripherally. The cytoplasm was pale and processes extended laterally or ramified among the surrounding thymocytes. This type of cell formed the majority of immunoreactive cells. The other cell type consisted of polygonal epithelial cells. The nucleus was oval with deep indentations. Euchromatin occupied a large part of the nucleus. The cytoplasm contained numerous cell organelles compared with the elongated type, in particular, electron-dense vacuoles of various sizes and often bundles of tonofilaments. Both types of epithelial cell were interconnected by desmosomes. No secretory granules were found in the cytoplasm of elongated or polygonal cells. These results indicate the presence of heterogeneous group of parvalbumin-immunoreactive epithelial cells and suggest the likelihood of different functional roles for parvalbumin in the pigeon thymus. PMID:10853953

  3. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  4. Evidence of apoptotic cell death after experimental traumatic brain injury in the rat.

    PubMed Central

    Rink, A.; Fung, K. M.; Trojanowski, J. Q.; Lee, V. M.; Neugebauer, E.; McIntosh, T. K.

    1995-01-01

    Apoptosis plays an important role in many developmental and pathological processes of the central nervous system. However, the role of apoptosis in traumatic brain injury has not been determined. Using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) method, we detected many cells with extensive DNA fragmentation in different regions of the brains of rats subjected to experimental traumatic brain injury. Two types of TUNEL-positive cells were demonstrated by light and electron microscopy, including type I cells that displayed morphological features of necrotic cell death and type II cells that displayed morphological features of classic apoptotic cell death. TUNEL-positive cells were detectable for up to 72 hours after the initial injury. Gel electrophoresis of DNA extracted from affected areas of the injured brain containing both type I and II cells revealed only internucleosomal fragmentation at 185-bp intervals, a feature originally described in apoptotic cell death. These data suggest that apoptosis, in addition to necrotic cell death, occurs after traumatic brain injury, and that internucleosomal fragmentation of DNA may be associated with certain types of necrotic cell death. Images Figure 1 Figure 2 Figure 4 PMID:7495282

  5. Ocimum gratissimum Aqueous Extract Induces Apoptotic Signalling in Lung Adenocarcinoma Cell A549

    PubMed Central

    Chen, Han-Min; Lee, Mu-Jang; Kuo, Cheng-Yi; Tsai, Pei-Lin; Liu, Jer-Yuh; Kao, Shao-Hsuan

    2011-01-01

    Ocimum gratissimum (OG) is widely used as a traditional herb for its antibacterial activity in Taiwan. Recently, antitumor effect of OG on breast cancer cell is also reported; however, the effects of OG on human pulmonary adenocarcinoma cell A549 remain unclear. Therefore, we aimed to investigate whether aqueous OG extract (OGE) affects viability of A549 cells and the signals induced by OGE in A549 cells. Cell viability assays revealed that OGE significantly and dose-dependently decreased the viability of A549 cell but not that of BEAS-2B cell. Morphological examination and DAPI staining indicated that OGE induced cell shrinkage and DNA condensation for A549 cells. Further investigation showed that OGE enhanced activation of caspase-3, caspase-9 and caspase-8 and increased protein level of Apaf-1 and Bak, but diminished the level of Bcl-2. Additionally, OGE inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) yet enhanced the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38). In conclusion, our findings indicate that OGE suppressed the cell viability of A549 cells, which may result from the activation of apoptotic signaling and the inhibition of anti-apoptotic signaling, suggesting that OGE might be beneficial to lung carcinoma treatment. PMID:20953389

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

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

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

  7. NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis.

    PubMed

    Chung, H; Vilaysane, A; Lau, A; Stahl, M; Morampudi, V; Bondzi-Simpson, A; Platnich, J M; Bracey, N A; French, M-C; Beck, P L; Chun, J; Vallance, B A; Muruve, D A

    2016-08-01

    Nod-like receptor, pyrin containing 3 (NLRP3) is characterized primarily as a canonical caspase-1 activating inflammasome in macrophages. NLRP3 is also expressed in the epithelium of the kidney and gut; however, its function remains largely undefined. Primary mouse tubular epithelial cells (TEC) lacking Nlrp3 displayed reduced apoptosis downstream of the tumor necrosis factor (TNF) receptor and CD95. TECs were identified as type II apoptotic cells that activated caspase-8, tBid and mitochondrial apoptosis via caspase-9, responses that were reduced in Nlrp3-/- cells. The activation of caspase-8 during extrinsic apoptosis induced by TNFα/cycloheximide (TNFα/CHX) was dependent on adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and completely independent of caspase-1 or caspase-11. TECs and primary human proximal tubular epithelial cells (HPTC) did not activate a canonical inflammasome, caspase-1, or IL-1β secretion in response to TNFα/CHX or NLRP3-dependent triggers, such as ATP or nigericin. In cell fractionation studies and by confocal microscopy, NLRP3 colocalized with ASC and caspase-8 in speck-like complexes at the mitochondria during apoptosis. The formation of NLRP3/ASC/caspase-8 specks in response to TNFα/CHX was downstream of TNFR signaling and dependent on potassium efflux. Epithelial ASC specks were present in enteroids undergoing apoptosis and in the injured tubules of wild-type but not Nlrp3-/- or ASC-/- mice following ureteric unilateral obstruction in vivo. These data show that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells. PMID:26891693

  8. Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA{sub 2} activity

    SciTech Connect

    Takatani-Nakase, Tomoka Takahashi, Koichi

    2015-07-17

    Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA{sub 2}, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death. - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA{sub 2} activity, leading to avoidance of non-apoptotic cell death.

  9. Proposed Pharmacological Countermeasures Against Apoptotic Cell Death in Experimental Models Mimicking Space Environment Damage

    NASA Astrophysics Data System (ADS)

    Lulli, Matteo; Papucci, Laura; Witort, Ewa; Donnini, Martino; Lapucci, Andrea; Lazzarano, Stefano; Mazzoni, Tiziano; Simoncini, Madine; Falciani, Piergiuseppe; Capaccioli, Sergio

    2008-06-01

    Several damaging agents have been suggested to affect human vision during long term space travels. Recently, apoptosis induced by DNA-damaging agents has emerged as frequent pathogenetic mechanism of ophthalmologic pathologies. Here, we propose two countermeasures: coenzyme Q10 and bcl-2 downregulation preventing antisense oligoribonucleotides (ORNs), aimed to inhibit cellular apoptotic death. Our studies have been carried out on retina and neuronal cultured cells treated with the following apoptotic stimuli mimicking space environment: a several-day exposure to either 3H-labeled tymidine or to the genotoxic drug doxorubicin, UV irradiation, hypoxia and glucose/growth factor starvation (Locke medium). The preliminary results clearly indicate that CoQ10, as well as bcl-2 down-regulation preventing ORNs, significantly counteract apoptosis in response to different DNA damaging agents in cultured eye and in neuronal cells. This supports the possibility that both could be optimal countermeasures against ophthalmologic lesions during space explorations.

  10. Cell volume regulation in epithelial physiology and cancer

    PubMed Central

    Pedersen, Stine F.; Hoffmann, Else K.; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered 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 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. PMID:24009588

  11. The Blockade of IL6 Counterparts the Osmolar Stress-Induced Apoptosis in Human Conjunctival Epithelial Cells

    PubMed Central

    Ju, Hee-Jung; Byun, Yong-Soo; Mok, Jee-Won

    2016-01-01

    To determine the effect of hyperosmolarity on cell survival/apoptosis of conjunctival epithelial cells and evaluate the possible role of IL6, Wong-Kilbourne derivative of Chang conjunctival cell line (WKD) was used in this study. Confluent cells were incubated under different osmolarity (290 mOsm and 500 mOsm) with or without neutralizing IL6 antibody (50 ng/mL). The expression of IL6 level was measured in the supernatant of each conditioned medium. Cell viability/apoptosis assay was performed using Annexin V/Propidium Iodide (PI) and Cell Counting Kit-8 (CCK-8). Western blot was conducted to measure the abundance of apoptotic markers and IL6 related downstream signaling pathway. The concentration of IL6 showed time-dependent increase in cells treated with 500 mOsm. Although apoptosis of WKD cell is increased in treated 500 mOsm for 24 h, apoptosis reduced in WKD cell treated 500 mOsm with anti-IL6 for 24 h. Anti-IL6 inhibited the activation of JAK-STAT signaling pathway, which was induced by hyperosmolarity. Hyperosmolar condition induced apoptosis in conjunctival epithelial cells, along with increase of IL6 production. IL6 neutralizing antibody inhibited apoptosis and JAK-STAT signaling in hyperosmolar condition. These findings suggested that IL6 may be involved in apoptotic change and in hyperosmolarity. PMID:27555966

  12. MFGE8 regulates TGF-β-induced epithelial mesenchymal transition in endometrial epithelial cells in vitro.

    PubMed

    Yu, Liang; Hu, Rong; Sullivan, Claretta; Swanson, R James; Oehninger, Sergio; Sun, Ying-Pu; Bocca, Silvina

    2016-09-01

    This study investigated the role of milk fat globule-epidermal growth factor-factor 8 (MFGE8) in TGF-β-induced epithelial-mesenchymal transition (EMT) of endometrial epithelial cells. These were in vitro studies using human endometrial epithelial cells and mouse blastocysts. We investigated the ability of TGF-β to induce EMT in endometrial epithelial cells (HEC-1A) by assessment of cytological phenotype (by light and atomic force microscopy), changes in expression of the markers of cell adhesion/differentiation E- and N-cadherin, and of the transcription factor Snail (by immunofluorescence and immunoblotting), and competence to support embryo attachment in a mouse blastocyst outgrowth assay. We also studied the effects of E-cadherin expression in cells transfected by retroviral shRNA vectors specifically silencing MFGE8. Results demonstrated that TGF-β induced EMT as demonstrated by phenotypic cell changes, by a switch of cadherin expression as well as by upregulation of the expression of the mesenchymal markers Snail and Vimentin. Upon MFGE8 knockdown, these processes were interfered with, suggesting that MFGE8 and TGF-β together may participate in regulation of EMT. This study demonstrated for the first time that endometrial MFGE8 modulates TGF-β-induced EMT in human endometrium cells. PMID:27340235

  13. Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells.

    PubMed

    Tian, K Y; Liu, X J; Xu, J D; Deng, L J; Wang, G

    2015-05-01

    Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (Δψm) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, Δψm reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway. PMID:25760023

  14. Morphological appearances of human lens epithelial cells in culture.

    PubMed

    Power, W; Neylan, D; Collum, L

    1993-01-01

    A system for culturing human lens epithelial cells in the laboratory was developed. The morphological appearances of the cells was studied using phase contrast, scanning and transmission electron microscopy. Cell marker studies using monoclonal antibodies to cytokeratin, vimentin and epithelial membrane antigen were also performed. There was a marked increase in cell size as a function of time in culture. After 3 to 4 weeks cells showed early signs of ageing. By 6 to 8 weeks the majority of the cells had become very irregular in shape and demonstrated irregularities of the plasma membrane and intra-cytoplasmic vacuole formation. The cells stained strongly for vimentin and epithelial membrane antigen. Staining with cytokeratin was somewhat weaker. This culture technique provides us with a suitable model for studying the growth behavior of these cells. PMID:7512459

  15. Regulated Mucin Secretion from Airway Epithelial Cells

    PubMed Central

    Adler, Kenneth B.; Tuvim, Michael J.; Dickey, Burton F.

    2013-01-01

    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 3 × 106 Da 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 μm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, 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

  16. Effect of freezing on lens epithelial cell growth.

    PubMed

    Fukaya, Y; Hara, T; Hara, T; Iwata, S

    1988-05-01

    The effect of freezing on the growth of rat lens epithelial cells was studied in vitro. We found that 80% of the lens epithelial cells died after freezing at -45 degrees C for two hours and that the surviving cells could grow with the addition of growth factors or when placed on a sheet of type 4 collagen, but not when placed on a plain plastic culture dish. These results suggest that the surviving cells are at the Go phase of the cell cycle and that type 4 collagen or growth factors can initiate cell division. PMID:3294380

  17. Hydrogen sulfide induces apoptosis in epithelial cells derived from human gingiva.

    PubMed

    Murata, T; Yaegaki, K; Qian, W; Herai, M; Calenic, B; Imai, T; Sato, T; Tanaka, T; Kamoda, T; Ii, H

    2008-03-01

    Hydrogen sulfide (H(2)S) is not only one of the main causes of halitosis but is also an agent of toxicity against periodontal cells and tissues in biofilm-related periodontal diseases. Also, apoptosis of gingival epithelial cells may play an important role in the onset and progress of periodontitis. We examined the effect of H(2)S on the induction of apoptosis, using human gingival fibroblasts (HGF) and keratinocyte-like Ca9-22 cells derived from human gingiva. The cells were incubated with H(2)S (100 ng ml(-1)) for 24, 48 or 72 h by adding H(2)S to air containing 5% CO(2), supplied constantly to the culture environment during incubation. The incidence of apoptosis caused by H(2)S was determined with Annexin V staining by flow cytometry. The proportion of apoptotic cells was significantly increased by exposure to H(2)S for 48 h in comparison with the control in both Ca9-22 cells and HGF. A concentration of 100 ng ml(-1) H(2)S in air is possible in the gingival sulcus. This study indicates that apoptosis in gingival epithelial cells and HGF by H(2)S may occur in the oral cavity, which may cause a periodontal condition. PMID:21386151

  18. How Shigella Utilizes Ca2+ Jagged Edge Signals during Invasion of Epithelial Cells

    PubMed Central

    Bonnet, Mariette; Tran Van Nhieu, Guy

    2016-01-01

    Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca2+ responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca2+ increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca2+ overload. In this review, we will focus on the role of Ca2+ responses and their regulation by Shigella during the different stages of bacterial infection. PMID:26904514

  19. How Shigella Utilizes Ca(2+) Jagged Edge Signals during Invasion of Epithelial Cells.

    PubMed

    Bonnet, Mariette; Tran Van Nhieu, Guy

    2016-01-01

    Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca(2+) responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca(2+) increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca(2+) overload. In this review, we will focus on the role of Ca(2+) responses and their regulation by Shigella during the different stages of bacterial infection. PMID:26904514

  20. Cell cycle regulation and apoptotic cell death in experimental colon carcinogenesis: intervening with cyclooxygenase-2 inhibitors.

    PubMed

    Saini, Manpreet Kaur; Sanyal, Sankar Nath

    2015-01-01

    Relative imbalance in the pathways regulating cell cycle, cell proliferation, or cell death marks a prerequisite for neoplasm. C-phycocyanin, a biliprotein from Spirulina platensis and a selective COX-2 inhibitor along with piroxicam, a traditional nonsteroidal antiinflammatory drug was used to investigate the role of cell cycle regulatory proteins and proinflammatory transcription factor NFκB in 1,2-dimethylhydrazine dihydrochloride (DMH)-induced rat colon carcinogenesis. Cell cycle regulators [cyclin D1, cyclin E, cyclin dependent kinase 2 (CDK2), CDK4, and p53], NFκB (p65) pathway, and proliferating cell nuclear antigen (PCNA) were evaluated by gene and protein expression, whereas apoptosis was studied by terminal deoxynucleotidyl transferase dUTP nick end labeling and apoptotic bleb assay. Molecular docking of ligand protein interaction was done to validate the in vivo results. Cyclin D1, cyclin E, CDK2, and CDK4 were overexpressed in DMH, whereas piroxicam and c-phycocyanin promoted the cell cycle arrest by downregulating them. Both drugs mediated apoptosis through p53 activation. Piroxicam and c-phycocyanin also stimulated antiproliferation by restraining PCNA expression and reduced cell survival via inhibiting NFκB (p65) pathway. Molecular docking revealed that phycocyanobilin (a chromophore of c-phycocyanin) interact with DNA binding site of NFκB. Inhibition of cyclin/CDK complex by piroxicam and c-phycocyanin affects the expression of p53 in colon cancer followed by downregulation of NFκB and PCNA levels, thus substantiating the antineoplastic role of these agents. PMID:25825916

  1. Anti-apoptotic peptides protect against radiation-induced cell death.

    PubMed

    McConnell, Kevin W; Muenzer, Jared T; Chang, Kathy C; Davis, Chris G; McDunn, Jonathan E; Coopersmith, Craig M; Hilliard, Carolyn A; Hotchkiss, Richard S; Grigsby, Perry W; Hunt, Clayton R

    2007-04-01

    The risk of terrorist attacks utilizing either nuclear or radiological weapons has raised concerns about the current lack of effective radioprotectants. Here it is demonstrated that the BH4 peptide domain of the anti-apoptotic protein Bcl-xL can be delivered to cells by covalent attachment to the TAT peptide transduction domain (TAT-BH4) and provide protection in vitro and in vivo from radiation-induced apoptotic cell death. Isolated human lymphocytes treated with TAT-BH4 were protected against apoptosis following exposure to 15Gy radiation. In mice exposed to 5Gy radiation, TAT-BH4 treatment protected splenocytes and thymocytes from radiation-induced apoptotic cell death. Most importantly, in vivo radiation protection was observed in mice whether TAT-BH4 treatment was given prior to or after irradiation. Thus, by targeting steps within the apoptosis signaling pathway it is possible to develop post-exposure treatments to protect radio-sensitive tissues. PMID:17307150

  2. ONCOGENE ALTERNATIONS IN IN VITRO TRANSFORMED RAT TRACHEAL EPITHELIAL CELLS

    EPA Science Inventory

    Ten derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 non-tumorigenic cell line transformed by treatment with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP) and/or 12-0-tetradecanoylphor...

  3. Apoptotic effects on cultured cells of atmospheric-pressure plasma produced using various gases

    NASA Astrophysics Data System (ADS)

    Tominami, Kanako; Kanetaka, Hiroyasu; Kudo, Tada-aki; Sasaki, Shota; Kaneko, Toshiro

    2016-01-01

    This study investigated the effects of low-temperature atmospheric-pressure plasma on various cells such as rat fibroblastic Rat-1 cell line, rat neuroblastoma-like PC12 cell line, and rat macrophage-like NR8383 cell line. The plasma was irradiated directly to a culture medium containing plated cells for 0-20 s. The applied voltage, excitation frequency, and argon or helium gas flow were, respectively, 3-6 kV, 10 kHz, and 3 L/min. Cell viability and apoptotic activity were evaluated using annexin-V/propidium iodide staining. Results showed that the low-temperature atmospheric-pressure plasma irradiation promoted cell death in a discharge-voltage-dependent and irradiation-time-dependent manner. Furthermore, different effects are produced depending on the cell type. Moreover, entirely different mechanisms might be responsible for the induction of apoptosis in cells by helium and argon plasma.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  5. Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

    SciTech Connect

    Nicolaou, Katerina A.; Liapis, Vasilis; Evdokiou, Andreas; Constantinou, Constantina; Magiatis, Prokopios; Skaltsounis, Alex L.; Koumas, Laura; Costeas, Paul A.; Constantinou, Andreas I.

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer The effects of 6BIO and 7BIO are evaluated against five breast cancer cell lines. Black-Right-Pointing-Pointer 6BIO induces a caspase dependent apoptotic effect via the intrinsic pathway. Black-Right-Pointing-Pointer 7BIO promotes G{sub 2}/M cells cycle arrest. Black-Right-Pointing-Pointer 7BIO triggers a caspase-8 mediated apoptotic pathway. Black-Right-Pointing-Pointer 7BIO triggers and a caspase independent pathway. -- Abstract: Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3 Prime -oxime (6BIO) and 7-bromoindirubin-3 Prime -oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G{sub 2}/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics.

  6. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

    PubMed Central

    Maggio, Savannah; Takeda, Kazuyo; Stark, Felicity; Meierovics, Anda I.; Yabe, Idalia; Cowley, Siobhan C.

    2015-01-01

    The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates. PMID:26379269

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

    SciTech Connect

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

    1997-10-13

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

  8. Interferons Mediate Terminal Differentiation of Human Cortical Thymic Epithelial Cells

    PubMed Central

    Vidalain, Pierre-Olivier; Laine, David; Zaffran, Yona; Azocar, Olga; Servet-Delprat, Christine; Wild, T. Fabian; Rabourdin-Combe, Chantal; Valentin, Hélène

    2002-01-01

    In the thymus, epithelial cells comprise a heterogeneous population required for the generation of functional T lymphocytes, suggesting that thymic epithelium disruption by viruses may compromise T-cell lymphopoiesis in this organ. In a previous report, we demonstrated that in vitro, measles virus induced differentiation of cortical thymic epithelial cells as characterized by (i) cell growth arrest, (ii) morphological and phenotypic changes, and (iii) apoptotis as a final step of this process. In the present report, we have analyzed the mechanisms involved. First, measles virus-induced differentiation of thymic epithelial cells is shown to be strictly dependent on beta interferon (IFN-β) secretion. In addition, transfection with double-stranded RNA, a common intermediate of replication for a broad spectrum of viruses, is reported to similarly mediate thymic epithelial cell differentiation through IFN-β induction. Finally, we demonstrated that recombinant IFN-α, IFN-β, or IFN-γ was sufficient to induce differentiation and apoptosis of uninfected thymic epithelial cells. These observations suggested that interferon secretion by either infected cells or activated leukocytes, such as plasmacytoid dendritic cells or lymphocytes, may induce thymic epithelium disruption in a pathological context. Thus, we have identified a new mechanism that may contribute to thymic atrophy and altered T-cell lymphopoiesis associated with many infections. PMID:12050353

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

    SciTech Connect

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

    1991-02-01

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

  10. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway.

    PubMed

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-01-01

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release 'messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial 'apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50-120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  11. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway

    PubMed Central

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-01-01

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release ‘messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelialapoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50–120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  12. Normal Cellular Prion Protein Protects against Manganese-induced Oxidative Stress and Apoptotic Cell Death

    PubMed Central

    Choi, Christopher J.; Anantharam, Vellareddy; Saetveit, Nathan J.; Houk, Robert. S.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2012-01-01

    The normal prion protein is abundantly expressed in the CNS, but its biological function remains unclear. The prion protein has octapeptide repeat regions that bind to several divalent metals, suggesting that the prion proteins may alter the toxic effect of environmental neurotoxic metals. In the present study, we systematically examined whether prion protein modifies the neurotoxicity of manganese (Mn) by comparing the effect of Mn on mouse neural cells expressing prion protein (PrPC -cells) and prion-knockout (PrPKO -cells). Exposure to Mn (10 μM-1 mM) for 24 hr produced a dose-dependent cytotoxic response in both PrPC -cells and PrPKO -cells. Interestingly, PrPC -cells (EC50 117.6μM) were more resistant to Mn-induced cytotoxicity, as compared to PrPKO -cells (EC50 59.9μM), suggesting a protective role for PrPC against Mn neurotoxicity. Analysis of intracellular Mn levels showed less Mn accumulation in PrPC -cells as compared to PrPKO -cells. Furthermore, Mn-induced mitochondrial depolarization and ROS generation were significantly attenuated in PrPC -cells as compared to PrPKO -cells. Measurement of antioxidant status revealed similar basal levels of glutathione (GSH) in PrPC -cells and PrPKO -cells; however, Mn treatment caused greater depletion of GSH in PrPKO -cells. Mn-induced mitochondrial depolarization and ROS production were followed by time- and dose-dependent activation of the apoptotic cell death cascade involving caspase-9 and -3. Notably, DNA fragmentation induced by both Mn treatment and oxidative stress-inducer hydrogen peroxide (100μM) was significantly suppressed in PrPC -cells as compared to PrPKO -cells. Together, these results demonstrate that prion protein interferes with divalent metal Mn uptake and protects against Mn-induced oxidative stress and apoptotic cell death. PMID:17483122

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

    PubMed

    Nighot, Prashant; Ma, Thomas

    2016-01-01

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

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

    PubMed

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

    2010-12-01

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

  15. The Epithelial Cell in Lung Health and Emphysema Pathogenesis

    PubMed Central

    Mercer, Becky A.; Lemaître, Vincent; Powell, Charles A.; D’Armiento, Jeanine

    2009-01-01

    Cigarette smoking is the primary cause of the irreversible lung disease emphysema. Historically, inflammatory cells such as macrophages and neutrophils have been studied for their role in emphysema pathology. However, recent studies indicate that the lung epithelium is an active participant in emphysema pathogenesis and plays a critical role in the lung’s response to cigarette smoke. Tobacco smoke increases protease production and alters cytokine expression in isolated epithelial cells, suggesting that these cells respond potently even in the absence of a complete inflammatory program. Tobacco smoke also acts as an immunosuppressant, reducing the defense function of airway epithelial cells and enhancing colonization of the lower airways. Thus, the paradigm that emphysema is strictly an inflammatory-cell based disease is shifting to consider the involvement of resident epithelial cells. Here we review the role of epithelial cells in lung development and emphysema. To better understand tobacco-epithelial interactions we performed microarray analyses of RNA from human airway epithelial cells exposed to smoke extract for 24 hours. These studies identified differential regulation of 425 genes involved in diverse biological processes, such as apoptosis, immune function, cell cycle, signal transduction, proliferation, and antioxidants. Some of these genes, including VEGF, glutathione peroxidase, IL-13 receptor, and cytochrome P450, have been previously reported to be altered in the lungs of smokers. Others, such as pirin, cathepsin L, STAT1, and BMP2, are shown here for the first time to have a potential role in smoke-associated injury. These data broaden our understanding of the importance of epithelial cells in lung health and cigarette smoke-induced emphysema. PMID:19662102

  16. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

    PubMed Central

    Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

    2002-01-01

    The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by

  17. The amino acid sensor GCN2 inhibits inflammatory responses to apoptotic cells promoting tolerance and suppressing systemic autoimmunity.

    PubMed

    Ravishankar, Buvana; Liu, Haiyun; Shinde, Rahul; Chaudhary, Kapil; Xiao, Wei; Bradley, Jillian; Koritzinsky, Marianne; Madaio, Michael P; McGaha, Tracy L

    2015-08-25

    Efficient apoptotic cell clearance and induction of immunologic tolerance is a critical mechanism preventing autoimmunity and associated pathology. Our laboratory has reported that apoptotic cells induce tolerance by a mechanism dependent on the tryptophan catabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) in splenic macrophages (MΦ). The metabolic-stress sensing protein kinase GCN2 is a primary downstream effector of IDO1; thus, we tested its role in apoptotic cell-driven immune suppression. In vitro, expression of IDO1 in MΦs significantly enhanced apoptotic cell-driven IL-10 and suppressed IL-12 production in a GCN2-dependent mechanism. Suppression of IL-12 protein production was due to attenuation of IL-12 mRNA association with polyribosomes inhibiting translation while IL-10 mRNA association with polyribosomes was not affected. In vivo, apoptotic cell challenge drove a rapid, GCN2-dependent stress response in splenic MΦs with increased IL-10 and TGF-β production, whereas myeloid-specific deletion of GCN2 abrogated regulatory cytokine production with provocation of inflammatory T-cell responses to apoptotic cell antigens and failure of long-tolerance induction. Consistent with a role in prevention of apoptotic cell driven autoreactivity, myeloid deletion of GCN2 in lupus-prone mice resulted in increased immune cell activation, humoral autoimmunity, renal pathology, and mortality. In contrast, activation of GCN2 with an agonist significantly reduced anti-DNA autoantibodies and protected mice from disease. Thus, this study implicates a key role for GCN2 signals in regulating the tolerogenic response to apoptotic cells and limiting autoimmunity. PMID:26261340

  18. The amino acid sensor GCN2 inhibits inflammatory responses to apoptotic cells promoting tolerance and suppressing systemic autoimmunity

    PubMed Central

    Ravishankar, Buvana; Liu, Haiyun; Shinde, Rahul; Chaudhary, Kapil; Xiao, Wei; Bradley, Jillian; Koritzinsky, Marianne; Madaio, Michael P.; McGaha, Tracy L.

    2015-01-01

    Efficient apoptotic cell clearance and induction of immunologic tolerance is a critical mechanism preventing autoimmunity and associated pathology. Our laboratory has reported that apoptotic cells induce tolerance by a mechanism dependent on the tryptophan catabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) in splenic macrophages (MΦ). The metabolic-stress sensing protein kinase GCN2 is a primary downstream effector of IDO1; thus, we tested its role in apoptotic cell-driven immune suppression. In vitro, expression of IDO1 in MΦs significantly enhanced apoptotic cell-driven IL-10 and suppressed IL-12 production in a GCN2-dependent mechanism. Suppression of IL-12 protein production was due to attenuation of IL-12 mRNA association with polyribosomes inhibiting translation while IL-10 mRNA association with polyribosomes was not affected. In vivo, apoptotic cell challenge drove a rapid, GCN2-dependent stress response in splenic MΦs with increased IL-10 and TGF-β production, whereas myeloid-specific deletion of GCN2 abrogated regulatory cytokine production with provocation of inflammatory T-cell responses to apoptotic cell antigens and failure of long-tolerance induction. Consistent with a role in prevention of apoptotic cell driven autoreactivity, myeloid deletion of GCN2 in lupus-prone mice resulted in increased immune cell activation, humoral autoimmunity, renal pathology, and mortality. In contrast, activation of GCN2 with an agonist significantly reduced anti-DNA autoantibodies and protected mice from disease. Thus, this study implicates a key role for GCN2 signals in regulating the tolerogenic response to apoptotic cells and limiting autoimmunity. PMID:26261340

  19. Phototoxic aptamers selectively enter and kill epithelial cancer cells

    PubMed Central

    Ferreira, Cátia S. M.; Cheung, Melissa C.; Missailidis, Sotiris; Bisland, Stuart; Gariépy, Jean

    2009-01-01

    The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells. PMID:19103663

  20. Protective natural autoantibodies to apoptotic cells: evidence of convergent selection of recurrent innate-like clones.

    PubMed

    Silverman, Gregg J

    2015-12-01

    During murine immune development, recurrent B cell clones arise in a predictable fashion. Among these B cells, an archetypical clonotypic set that recognizes phosphorylcholine (PC) antigens and produces anti-PC IgM, first implicated for roles in microbial protection, was later found to become expanded in hyperlipidemic mice and in response to an increased in vivo burden of apoptotic cells. These IgM natural antibodies can enhance clearance of damaged cells and induce intracellular blockade of inflammatory signaling cascades. In clinical populations, raised levels of anti-PC IgM correlate with protection from atherosclerosis and may also downmodulate the severity of autoimmune disease. Human anti-PC-producing clones without hypermutation have been isolated that can similarly discriminate apoptotic from healthy cells. An independent report on unrelated adults has described anti-PC-producing B cells with IgM genes that have conserved CDR3 motifs, similar to stereotypic clonal sets of B cell chronic lymphocytic leukemia. Taken together, emerging evidence suggests that, despite the capacity to form an effectively limitless range of Ig receptors, the human immune system may often recurrently generate lymphocytes expressing structurally convergent B cell receptors with protective and homeostatic roles. PMID:25990717

  1. Galectin-1 and Galectin-3 induce mitochondrial apoptotic pathway in Jurkat cells

    NASA Astrophysics Data System (ADS)

    Vasil'eva, O. A.; Isaeva, A. V.; Prokhorenko, T. S.; Zima, A. P.; Novitsky, V. V.

    2016-08-01

    Cellular malignant transformation is often accompanied by increased gene expression of low-molecular proteins of lectins family-galectins. But it is unknown how galectins promote tumor growth and malignization. Galectins-1 and galectin-3 are thought to be possible immunoregulators exerting their effects by regulating the balance of CD4+ lymphocytes. In addition it is known that tumor cells overexpressing galectins are capable of escaping immunological control, causing apoptosis of lymphocytes. The aim of the study is to investigate the role of galectin-1 and galectin-3 in the implementation of mitochondrial apoptotic pathway in Jurkat cells. Methods: Jurkat cells were used as a model for the study of T-lymphocytes. Jurkat cells were activated with antibodies to CD3 and CD28 and cultured with recombinant galectin-1 and -3. Apoptosis of Jurkat cells and depolarization of the mitochondrial membrane were assessed by flow cytometry. It was found that galectin-1 and galectin-3 have a dose-dependent pro-apoptotic effect on Jurkat cells in vitro and enlarge the number of cells with decreased mitochondrial membrane potential compared with intact cells.

  2. MUC1 extracellular domain confers resistance of epithelial cancer cells to anoikis

    PubMed Central

    Zhao, Q; Piyush, T; Chen, C; Hollingsworth, M A; Hilkens, J; Rhodes, J M; Yu, L-G

    2014-01-01

    Anoikis, a special apoptotic process occurring in response to loss of cell adhesion to the extracellular matrix, is a fundamental surveillance process for maintaining tissue homeostasis. Resistance to anoikis characterises cancer cells and is a pre-requisite for metastasis. This study shows that overexpression of the transmembrane mucin protein MUC1 prevents initiation of anoikis in epithelial cancer cells in response to loss of adhesion. We show that this effect is largely attributed to the elongated and heavily glycosylated extracellular domain of MUC1 that protrudes high above the cell membrane and hence prevents activation of the cell surface anoikis-initiating molecules such as integrins and death receptors by providing them a mechanically ‘homing' microenvironment. As overexpression of MUC1 is a common feature of epithelial cancers and as resistance to anoikis is a hallmark of both oncogenic epithelial–mesenchymal transition and metastasis, MUC1-mediated cell resistance to anoikis may represent one of the fundamental regulatory mechanisms in tumourigenesis and metastasis. PMID:25275599

  3. Differential interaction of bacterial species from the Burkholderia cepacia complex with human airway epithelial cells.

    PubMed

    Moura, Jane A; Cristina de Assis, Maria; Ventura, Grasiella C; Saliba, Alessandra M; Gonzaga, Luiz; Si-Tahar, Mustapha; Marques, Elizabeth de A; Plotkowski, Maria Cristina

    2008-01-01

    To increase knowledge of the pathogenic potential of the Burkholderia cepacia complex (BCC), we investigated the effects of reference strains of the nine BCC species on human bronchial epithelial cells in vitro. B. multivorans exhibited the highest rates of adherence to and internalization by host cells. Two out of three clinical isolates recovered from cystic fibrosis patients confirmed the B. multivorans high adhesiveness. All four B. multivorans isolates exhibited an aggregated pattern of adherence but any of them expressed cable pili. When bacteria were centrifuged onto cell cultures to circumvent their poor adhesiveness, B. pyrrocinia exhibited the highest internalization rate, followed by B. multivorans. The percentages of apoptotic cells in cultures infected with B. cepacia, B. multivorans, B. cenocepacia (subgroups IIIA and IIIB), B. stabilis and B. vietnamiensis were significantly higher than in control non-infected cultures. All nine BCC species triggered a similar release of the inflammatory cytokine IL-8, that was not reduced by cell treatment with cytochalasin D. Hence, our data demonstrate, for the first time, that all BCC species exhibit a similar ability to induce the expression of host immune mediators whereas they differ on their ability to adhere to, invade and kill airway epithelial cells. PMID:18068390

  4. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

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

    2012-09-14

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

  5. Distinct Modes of Macrophage Recognition for Apoptotic and Necrotic Cells Are Not Specified Exclusively by Phosphatidylserine Exposure

    PubMed Central

    Cocco, Regina E.; Ucker, David S.

    2001-01-01

    The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead cells. This clearance is believed to reveal an innate immunity, associated with inflammation in cases of pathological but not physiological cell deaths. Using objective and quantitative measures to assess these processes, we find that macrophages bind and engulf native apoptotic and necrotic cells to similar extents and with similar kinetics. However, recognition of these two classes of dying cells occurs via distinct and noncompeting mechanisms. Phosphatidylserine, which is externalized on both apoptotic and necrotic cells, is not a specific ligand for the recognition of either one. The distinct modes of recognition for these different corpses are linked to opposing responses from engulfing macrophages. Necrotic cells, when recognized, enhance proinflammatory responses of activated macrophages, although they are not sufficient to trigger macrophage activation. In marked contrast, apoptotic cells profoundly inhibit phlogistic macrophage responses; this represents a cell-associated, dominant-acting anti-inflammatory signaling activity acquired posttranslationally during the process of physiological cell death. PMID:11294896

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

    PubMed

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

    2015-11-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  7. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    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. PMID:26828484

  8. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

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

    1990-02-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  10. Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution.

    PubMed

    Hamazaki, Yoko; Sekai, Miho; Minato, Nagahiro

    2016-05-01

    The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed. PMID:27088906

  11. Sam68 is cleaved by caspases under apoptotic cell death induced by ionizing radiation.

    PubMed

    Cho, Seong-Jun; Choi, Moo Hyun; Nam, Seon Young; Kim, Ji Young; Kim, Cha Soon; Pyo, Suhkneung; Yang, Kwang Hee

    2015-03-01

    The RNA-binding protein Sam68, a mitotic substrate of tyrosine kinases, has been reported to participate in the cell cycle, apoptosis, and signaling. In particular, overexpression of Sam68 protein is known to suppress cell growth and cell cycle progression in NIH3T3 cells. Although Sam68 is involved in many cellular activities, the function of Sam68, especially in response to apoptotic stimulation, is not well understood. In this study, we found that Sam68 protein is cleaved in immune cells undergoing apoptosis induced by γ-radiation. Moreover, we found that Sam68 cleavage was induced by apoptotic stimuli containing γ-radiation in a caspase-dependent manner. In particular, we showed that activated casepase-3, 7, 8 and 9 can directly cleave Sam68 protein through in vitro protease cleavage assay. Finally, we found that the knockdown of Sam68 attenuated γ-radiation-induced cell death and growth suppression. Conclusively, the cleavage of Sam68 is a new indicator for the cell damaging effects of ionizing radiation. PMID:25666188

  12. Primula auriculata Extracts Exert Cytotoxic and Apoptotic Effects against HT-29 Human Colon Adenocarcinoma Cells.

    PubMed

    Behzad, Sahar; Ebrahim, Karim; Mosaddegh, Mahmoud; Haeri, Ali

    2016-01-01

    Primula auriculata (Tootia) is one of the most important local medicinal plants in Hamedan district, Iran. To investigate cytotoxicity and apoptosis induction of crude methanolic extract and different fraction of it, we compared several methods on HT-29 human colon Adenocarcinoma cells. Cancer cell proliferation was measured by 3-(4, 5‑dimethylthiazolyl)2, 5‑diphenyl‑tetrazolium bromide (MTT) assay and apoptosis induction was analyzed by fluorescence microscopy (acridin orange/ethidium bromide, annexin V/propidium iodide staining, TUNEL assay and Caspase-3 activity assay). Crude methanolic extract (CM) inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions, the dichloromethane fraction (CF) was found to be the most toxic compared to other fractions. With double staining methods, high percentage of 40 µg/mL of (CM) and (CF) treated cells exhibited typical characteristics of apoptotic cells. Apoptosis induction was also revealed by apoptotic fragmentation of nuclear DNA and activation of caspas-3 in treated cells. These findings indicate that crude methanolic extract and dichloromethan fraction of P.auriculata induced apoptosis and inhibited proliferation in colon cancer cells and could be used as a source for new lead structures in drug design to combat colon cancer. PMID:27610172

  13. Primula auriculata Extracts Exert Cytotoxic and Apoptotic Effects against HT-29 Human Colon Adenocarcinoma Cells

    PubMed Central

    Behzad, Sahar; Ebrahim, Karim; Mosaddegh, Mahmoud; Haeri, Ali

    2016-01-01

    Primula auriculata (Tootia) is one of the most important local medicinal plants in Hamedan district, Iran. To investigate cytotoxicity and apoptosis induction of crude methanolic extract and different fraction of it, we compared several methods on HT-29 human colon Adenocarcinoma cells. Cancer cell proliferation was measured by 3-(4, 5‑dimethylthiazolyl)2, 5‑diphenyl‑tetrazolium bromide (MTT) assay and apoptosis induction was analyzed by fluorescence microscopy (acridin orange/ethidium bromide, annexin V/propidium iodide staining, TUNEL assay and Caspase-3 activity assay). Crude methanolic extract (CM) inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions, the dichloromethane fraction (CF) was found to be the most toxic compared to other fractions. With double staining methods, high percentage of 40 µg/mL of (CM) and (CF) treated cells exhibited typical characteristics of apoptotic cells. Apoptosis induction was also revealed by apoptotic fragmentation of nuclear DNA and activation of caspas-3 in treated cells. These findings indicate that crude methanolic extract and dichloromethan fraction of P.auriculata induced apoptosis and inhibited proliferation in colon cancer cells and could be used as a source for new lead structures in drug design to combat colon cancer. PMID:27610172

  14. Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2) cell line

    PubMed Central

    2011-01-01

    Background Six plants from Thailand were evaluated for their cytotoxicity and apoptosis induction in human hepatocarcinoma (HepG2) as compared to normal African green monkey kidney epithelial cell lines. Methods Ethanol-water crude extracts of the six plants were tested with neutral red assay for their cytotoxicity after 24 hours of exposure to the cells. Apoptotic induction was tested in the HepG2 cells with diamidino-2-phenylindole staining. DNA fragmentation, indicative of apoptosis, was analyzed with agarose gel electrophoresis. Alkylation, indicative of DNA damage, was also evaluated in vitro by 4-(4'-nitrobenzyl) pyridine assay. Results The extract of Pinus kesiya showed the highest selectivity (selectivity index = 9.6) and potent cytotoxicity in the HepG2 cell line, with an IC50 value of 52.0 ± 5.8 μg/ml (mean ± standard deviation). Extract of Catimbium speciosum exerted cytotoxicity with an IC50 value of 55.7 ± 8.1 μg/ml. Crude extracts from Glochidion daltonii, Cladogynos orientalis, Acorus tatarinowii and Amomum villosum exhibited cytotoxicity with IC50 values ranging 100-500 μg/ml. All crude extracts showed different alkylating abilities in vitro. Extracts of P. kesiya, C. speciosum and C. orientalis caused nuclei morphological changes and DNA laddering. Conclusion The extracts of C. speciosum, C. orientalis and P. kesiya induced apoptosis. Among the three plants, P. kesiya possessed the most robust anticancer activity, with specific selectivity against HepG2 cells. PMID:22041055

  15. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets. PMID:23475606

  16. A NPxY-independent {beta}5 integrin activation signal regulates phagocytosis of apoptotic cells

    SciTech Connect

    Singh, Sukhwinder; D'mello, Veera; Henegouwen, Paul van Bergen en; Birge, Raymond B.

    2007-12-21

    Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the {beta} chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 ({beta}5) integrin cDNA was expressed in {alpha}v positive, {beta}5 and {beta}3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, {beta}5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing {beta}5 mutant (Y750A) abrogated adhesion and {beta}5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of {beta}5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a {beta}5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 {mu}m diameter microspheres developed as apoptotic cell mimetics. The critical sequences in {beta}5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of {beta}5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the {beta}5 cytoplasmic tail for adhesion and phagocytosis.

  17. Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

    PubMed Central

    2013-01-01

    Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with high clearance efficiency, thereby limiting cellularity and maintaining lesion integrity. However, as lesions mature, phagocytic removal of apoptotic cells (efferocytosis) becomes defective, leading to secondary necrosis, expansion of plaque necrotic cores, and susceptibility to rupture. Recent genetic causation studies in experimental rodents have implicated key molecular regulators of efferocytosis in atherosclerotic progression. These include MER tyrosine kinase (MERTK), milk fat globule-EGF factor 8 (MFGE8), and complement C1q. At the cellular level, atheromata are infiltrated by a heterogenous population of professional phagocytes, comprised of monocytes, differentiated macrophages, and CD11c+ dendritic-like cells. Each cell type is characterized by disparate clearance efficiencies and varying activities of key phagocytic signaling molecules. It is in this context that we outline a working model whereby plaque necrosis and destabilization is jointly promoted by (1) direct inhibition of core phagocytic signaling pathways and (2) expansion of phagocyte subsets with poor clearance capacity. Towards identifying targets for promoting efficient apoptotic cell clearance and resolving inflammation in atherosclerosis and during ischemic heart disease and post myocardial infarction, this review will discuss potential in vivo suppressors of efferocytosis at each stage of clearance and how these putative interventional targets may differentially affect uptake at the level of vascular phagocyte subsets. PMID:20552278

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

    PubMed Central

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

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. DOI: http://dx.doi.org/10.7554/eLife.15034.001 PMID:27431614

  19. CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

    PubMed Central

    Grek, Christina L.; Newton, Danforth A.; Qiu, Yonhzhi; Wen, Xuejun; Spyropoulos, Demetri D.; Baatz, John E.

    2012-01-01

    Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells. PMID:19263283

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

    PubMed Central

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

    2015-01-01

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

  1. Comparative in vitro cytotoxicity of modified deoxynivalenol on porcine intestinal epithelial cells.

    PubMed

    Broekaert, Nathan; Devreese, Mathias; Demeyere, Kristel; Berthiller, Franz; Michlmayr, Herbert; Varga, Elisabeth; Adam, Gerhard; Meyer, Evelyne; Croubels, Siska

    2016-09-01

    The gastrointestinal tract is the first target after ingestion of the mycotoxin deoxynivalenol (DON) via feed and food. Deoxynivalenol is known to affect the proliferation and viability of animal and human intestinal epithelial cells. In addition to DON, feed and food is often co-contaminated with modified forms of DON, such as 3-acetyldeoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) and deoxynivalenol-3-β-D-glucoside (DON3G). The goal of this study was to determine the in vitro intrinsic cytotoxicity of these modified forms towards differentiated and proliferative porcine intestinal epithelial cells by means of flow cytometry. Cell death was assessed by dual staining with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), which allows the discrimination of viable (FITC-/PI-), apoptotic (FITC+/PI-) and necrotic cells (FITC+/PI+). Based on the data from the presented pilot in vitro study, it is concluded that cytotoxicity for proliferative cells can be ranked as follows: DON3G ≪ 3ADON < DON≈15ADON. PMID:27338712

  2. Repression of SRF target genes is critical for Myc-dependent apoptosis of epithelial cells

    PubMed Central

    Wiese, Katrin E; Haikala, Heidi M; von Eyss, Björn; Wolf, Elmar; Esnault, Cyril; Rosenwald, Andreas; Treisman, Richard; Klefström, Juha; Eilers, Martin

    2015-01-01

    Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF-dependent transcriptional program that supports survival of epithelial cells. PMID:25896507

  3. Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions.

    PubMed

    Thangam, Ramar; Sathuvan, Malairaj; Poongodi, Arasu; Suresh, Veeraperumal; Pazhanichamy, Kalailingam; Sivasubramanian, Srinivasan; Kanipandian, Nagarajan; Ganesan, Nalini; Rengasamy, Ramasamy; Thirumurugan, Ramasamy; Kannan, Soundarapandian

    2014-07-17

    Essential oils of Cymbopogon citratus were already reported to have wide ranging medical and industrial applications. However, information on polysaccharides from the plant and their anticancer activities are limited. In the present study, polysaccharides from C. citratus were extracted and fractionated by anion exchange and gel filtration chromatography. Two different polysaccharide fractions such as F1 and F2 were obtained, and these fractions were found to have distinct acidic polysaccharides as characterized by their molecular weight and sugar content. NMR spectral analysis revealed the presence of (1→4) linked b-d-Xylofuranose moiety in these polysaccharides. Using these polysaccharide fractions F1 and F2, anti-inflammatory and anticancer activities were evaluated against cancer cells in vitro and the mechanism of action of the polysaccharides in inducing apoptosis in cancer cells via intrinsic pathway was also proposed. Two different reproductive cancer cells such as Siha and LNCap were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation, changes in mitochondrial membrane potential, and profiles of gene and protein expression in response to treatment of cells by the polysaccharide fractions. These polysaccharide fractions exhibited potential cytotoxic and apoptotic effects on carcinoma cells, and they induced apoptosis in these cells through the events of up-regulation of caspase 3, down-regulation of bcl-2 family genes followed by cytochrome c release. PMID:24702929

  4. BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module.

    PubMed

    Park, Daeho; Tosello-Trampont, Annie-Carole; Elliott, Michael R; Lu, Mingjian; Haney, Lisa B; Ma, Zhong; Klibanov, Alexander L; Mandell, James W; Ravichandran, Kodi S

    2007-11-15

    Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms. ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. However, the receptor(s) upstream of the ELMO/Dock180/Rac module are still unknown. Here we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells, as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells. PMID:17960134

  5. [Effect of bone marrow stromal cells on the apoptotic sensitivity of HL-60 and HL-60/VCR cells].

    PubMed

    Liang, Rong; Huang, Gao-Sheng; Wang, Zhe; Chen, Xie-Qun; Bai, Qing-Xian; Zhang, Wei-Ping; Wang, Juan-Hong; Wang, Wen-Qing; Guo, Ying

    2005-04-01

    This study was aimed to investigate the effects of human bone marrow fibroblastoid stromal cell line (HFCL) on chemosensitivity of acute myeloid leukemia sensitive HL-60 cell line and multidrug-resistant (MDR) HL-60/VCR cell line in vitro co-culture. Setting up co-culture system of HL-60 or HL-60/VCR cells in direct contact with HFCL cells, or with HFCL cells separated by transwell, and exposing HL-60 or HL-60/VCR cells to different concentrations of topotecon (TPT), morphologic evidence for apoptosis was determined by staining with Wright-Giemsa stain and acridine orange/ethidium bromide (AO/EB). Cell cycle, sub-G(1) and annexin V FITC staining were detected by flow cytometry. The expression of active caspase-3, Bcl-2 and Pgp was detected by Western blot. The results showed that HL-60 or HL-60/VCR cells treated by TPT revealed characteristic apoptotic morphological changes by Wright-Giemsa and AO/EB staining. The percentage of annexin V-positive cells and apoptotic cells decreased when they were cocultured with HFCL cells. The proportion of G(0)/G(1) HL-60 or HL-60/VCR cells treated by TPT increased and the sub-G(1) appeared significantly, but apoptotic and sub-G cells reduced after direct contact with HFCL cells. Meanwhile, although HL-60 or HL-60/VCR cells treated by TPT expressed activated caspase-3, and the expression of Bcl-2 decreased, the expression of activated caspase-3 decreased and Bcl-2 increased after direct contact with HFCL cells. In conclusion, HFCL stromal cells can prevent TPT-induced apoptosis in HL-60 and HL-60/VCR cells via modulation of Bcl-2 and active caspase-3. PMID:15854294

  6. Autoantibodies against complement C1q specifically target C1q bound on early apoptotic cells.

    PubMed

    Bigler, Cornelia; Schaller, Monica; Perahud, Iryna; Osthoff, Michael; Trendelenburg, Marten

    2009-09-01

    Autoantibodies against complement C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE). They strongly correlate with the occurrence of severe lupus nephritis, suggesting a pathogenic role in SLE. Because anti-C1q are known to recognize a neoepitope on bound C1q, but not on fluid-phase C1q, the aim of this study was to clarify the origin of anti-C1q by determining the mechanism that renders C1q antigenic. We investigated anti-C1q from serum and purified total IgG of patients with SLE and hypocomplementemic urticarial vasculitis as well as two monoclonal human anti-C1q Fab from a SLE patient generated by phage display. Binding characteristics, such as their ability to recognize C1q bound on different classes of Igs, on immune complexes, and on cells undergoing apoptosis, were analyzed. Interestingly, anti-C1q did not bind to C1q bound on Igs or immune complexes. Neither did we observe specific binding of anti-C1q to C1q bound on late apoptotic/necrotic cells when compared with binding in the absence of C1q. However, as shown by FACS analysis and confocal microscopy, anti-C1q specifically targeted C1q bound on early apoptotic cells. Anti-C1q were found to specifically target C1q bound on cells undergoing apoptosis. Our observations suggest that early apoptotic cells are a major target of the autoimmune response in SLE and provide a direct link between human SLE, apoptosis, and C1q. PMID:19648280

  7. Burkholderia pseudomallei Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells

    PubMed Central

    Kunyanee, Chanikarn; Kamjumphol, Watcharaporn; Taweechaisupapong, Suwimol; Kanthawong, Sakawrat; Wongwajana, Suwin; Wongratanacheewin, Surasak; Hahnvajanawong, Chariya

    2016-01-01

    Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses. PMID:27529172

  8. Investigation of the apoptotic pathway induced by benzimidazole-oxindole conjugates against human breast cancer cells MCF-7.

    PubMed

    Lakshma Nayak, Vadithe; Nagaseshadri, Bobburi; Vishnuvardhan, M V P S; Kamal, Ahmed

    2016-07-15

    In our previous studies, benzimidazole-oxindole conjugates were synthesized and evaluated by National Cancer Institute (NCI) for their cytotoxic activity and the new molecules like 5c and 5p were considered as potential leads. These conjugates arrested the cell cycle at G2/M phase and inhibited tubulin polymerization. These observations prompted us to investigate the apoptotic mechanism induced by these lead molecules against human breast cancer cells (MCF-7). Studies like measurement of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species (ROS) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis in human breast cancer cells. It was further confirmed by western blot analysis of pro apoptotic protein Bax, anti apoptotic protein Bcl-2, cytochrome c release, caspase-9 activity and cleavage of PARP. PMID:27262596

  9. USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death.

    PubMed

    Liang, Jin-Rui; Martinez, Aitor; Lane, Jon D; Mayor, Ugo; Clague, Michael J; Urbé, Sylvie

    2015-05-01

    Mitochondria play a pivotal role in the orchestration of cell death pathways. Here, we show that the control of ubiquitin dynamics at mitochondria contributes to the regulation of apoptotic cell death. The unique mitochondrial deubiquitylase, USP30, opposes Parkin-dependent ubiquitylation of TOM20, and its depletion enhances depolarization-induced cell death in Parkin-overexpressing cells. Importantly, USP30 also regulates BAX/BAK-dependent apoptosis, and its depletion sensitizes cancer cells to BH3-mimetics. These results provide the first evidence for a fundamental role of USP30 in determining the threshold for mitochondrial cell death and suggest USP30 as a potential target for combinatorial anti-cancer therapy. PMID:25739811

  10. USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death

    PubMed Central

    Liang, Jin-Rui; Martinez, Aitor; Lane, Jon D; Mayor, Ugo; Clague, Michael J; Urbé, Sylvie

    2015-01-01

    Mitochondria play a pivotal role in the orchestration of cell death pathways. Here, we show that the control of ubiquitin dynamics at mitochondria contributes to the regulation of apoptotic cell death. The unique mitochondrial deubiquitylase, USP30, opposes Parkin-dependent ubiquitylation of TOM20, and its depletion enhances depolarization-induced cell death in Parkin-overexpressing cells. Importantly, USP30 also regulates BAX/BAK-dependent apoptosis, and its depletion sensitizes cancer cells to BH3-mimetics. These results provide the first evidence for a fundamental role of USP30 in determining the threshold for mitochondrial cell death and suggest USP30 as a potential target for combinatorial anti-cancer therapy. PMID:25739811

  11. Distinction Between Cell Proliferation and Apoptosis Signals Regulated by Brain-Derived Neurotrophic Factor in Human Periodontal Ligament Cells and Gingival Epithelial Cells.

    PubMed

    Kashiwai, Kei; Kajiya, Mikihito; Matsuda, Shinji; Ouhara, Kazuhisa; Takeda, Katsuhiro; Takata, Takashi; Kitagawa, Masae; Fujita, Tsuyoshi; Shiba, Hideki; Kurihara, Hidemi

    2016-07-01

    Previously, we reported that brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration by inducing periodontal ligament cell proliferation in vivo. In addition, the down growth of gingival epithelial cells, which comprises a major obstacle to the regeneration, was not observed. However, the underlying molecular mechanism is still unclear. Therefore, this study aimed to investigate the effect of BDNF on cell proliferation and apoptosis in human periodontal ligament (HPL) cells and human gingival epithelial cells (OBA9 cells) and to explore the molecular mechanism in vitro. HPL cells dominantly expressed a BDNF receptor, TrkB, and BDNF increased cell proliferation and ERK phosphorylation. However, its proliferative effect was diminished by a MEK1/2 inhibitor (U0126) and TrkB siRNA transfection. Otherwise, OBA9 cells showed a higher expression level of p75, which is a pan-neurotrophin receptor, than that of HPL cells. BDNF facilitated not cell proliferation but cell apoptosis and JNK phosphorylation in OBA9 cells. A JNK inhibitor (SP600125) and p75 siRNA transfection attenuated the BDNF-induced cell apoptosis. Moreover, OBA9 cells pretreated with SP600125 or p75 siRNA showed cell proliferation by BDNF stimulation, though it was reduced by U0126 and TrkB siRNA. Interestingly, overexpression of p75 in HPL cells upregulated cell apoptosis and JNK phosphorylation by BDNF treatment. These results indicated that TrkB-ERK signaling regulates BDNF-induced cell proliferation, whereas p75-JNK signaling plays roles in cell apoptotic and cytostatic effect of BDNF. Overall, BDNF activates periodontal ligament cells proliferation and inhibits the gingival epithelial cells growth via the distinct pathway. J. Cell. Biochem. 117: 1543-1555, 2016. © 2015 Wiley Periodicals, Inc. PMID:26581032

  12. Nivalenol and Deoxynivalenol Affect Rat Intestinal Epithelial Cells: A Concentration Related Study

    PubMed Central

    Bianco, Giuseppe; Fontanella, Bianca; Severino, Lorella; Quaroni, Andrea; Autore, Giuseppina; Marzocco, Stefania

    2012-01-01

    The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5–80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G0/G1 interphase and in the S phase, with a concomitant reduction in the fraction of cells in G2. Interestingly, when administered at lower concentrations (0.1–2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins. PMID:23251682

  13. Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study.

    PubMed

    Bianco, Giuseppe; Fontanella, Bianca; Severino, Lorella; Quaroni, Andrea; Autore, Giuseppina; Marzocco, Stefania

    2012-01-01

    The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5-80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G(0)/G(1) interphase and in the S phase, with a concomitant reduction in the fraction of cells in G(2). Interestingly, when administered at lower concentrations (0.1-2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins. PMID:23251682

  14. Lymphotoxin beta receptor signaling limits mucosal damage through driving IL-23 production by epithelial cells.

    PubMed

    Macho-Fernandez, E; Koroleva, E P; Spencer, C M; Tighe, M; Torrado, E; Cooper, A M; Fu, Y-X; Tumanov, A V

    2015-03-01

    The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LTβR) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LTβR signaling in intestinal epithelial cells is essential for epithelial interleukin-23 (IL-23) production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4(-)CCR6(+)T-bet(-) RAR-related orphan receptor gamma t (RORγt)(+) lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LTβR signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage. PMID:25183367

  15. Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

    PubMed Central

    Ignatius, Ralf; Marovich, Mary; Mehlhop, Erin; Villamide, Loreley; Mahnke, Karsten; Cox, William I.; Isdell, Frank; Frankel, Sarah S.; Mascola, John R.; Steinman, Ralph M.; Pope, Melissa

    2000-01-01

    Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-α) secretion and was significantly blocked in the presence of anti-TNF-α antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-α and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-α secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors. PMID:11070033

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

    SciTech Connect

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

    2008-06-26

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

  17. Porphyromonas gingivalis invades oral epithelial cells in vitro.

    PubMed

    Sandros, J; Papapanou, P; Dahlén, G

    1993-05-01

    The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells. PMID:8388449

  18. Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Sharma, Ashu; Genco, Robert J.

    2002-01-01

    The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial

  19. Differentiation of porcine mesenchymal stem cells into epithelial cells as a potential therapeutic application to facilitate epithelial regeneration.

    PubMed

    Kokubun, Kelsey; Pankajakshan, Divya; Kim, Min-Jung; Agrawal, Devendra K

    2016-02-01

    Epithelial denudation is one of the characteristics of chronic asthma. To restore its functions, the airway epithelium has to rapidly repair the injuries and regenerate its structure and integrity. Mesenchymal stem cells (MSCs) have the ability to differentiate into many cell lineages. However, the differentiation of MSCs into epithelial cells has not been fully studied. Here, we examined the differentiation of MSCs into epithelial cells using three different media compositions with various growth supplementations. The MSCs were isolated from porcine bone marrow by density gradient centrifugation. The isolated MSCs were CD11(-) CD34(-) CD45(-) CD44(+) CD90(+) and CD105(+) by immunostaining and flow cytometry. MSCs were stimulated with EpiGRO (Millipore), BEpiCM (ScienCell) and AECGM (PromoCell) media for 5 and 10 days, and epithelial differentiation was assessed by qPCR (keratin 14, 18 and EpCAM), fluorometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM), western blot analysis (pancytokeratin, EpCAM) and flow cytometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM). The functional marker MUC1 was also assessed after 10 days of air-liquid interface (ALI) culture in optimized media. Cells cultured in BEpiCM containing fibroblast growth factor and prostaglandin E2 showed the highest expression of the epithelial markers: CK7-8 (85.90%); CK-14-15-16-19 (10.14%); and EpCAM (64.61%). The cells also expressed functional marker MUC1 after ALI culture. The differentiated MSCs when cultured in BEpiCM medium ex vivo in a bioreactor on a decellularized trachea for 10 days retained the epithelial-like phenotype. In conclusion, porcine bone marrow-derived MSCs demonstrate commitment to the epithelial lineage and might be a potential therapy for facilitating the repair of denuded airway epithelium. PMID:23696537

  20. Amniotic epithelial cells promote wound healing in mice through high epithelialization and engraftment.

    PubMed

    Jin, Enze; Kim, Tae-Hee; Han, Seongho; Kim, Sung-Whan

    2016-07-01

    Although human amniotic epithelial cells (AMEs) are an attractive source of stem cells, their therapeutic potential in wound healing has not been fully investigated. We evaluated the therapeutic potential of AMEs for wound healing. Real-time PCR showed that the epithelialization growth factors epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-B and chemotactic factors interleukin-8 (IL-8 or CXCL8) and neutrophil-activating protein-2 (NAP-2 or CXCL7) were upregulated in AMEs compared with adipose-derived mesenchymal stem cells (ADMs). In vitro scratch wound assays revealed that AME-derived conditioned medium substantially accelerated wound closure. Wounds in NOD/SCID mice were created by skin excision, followed by AME transplantation. AMEs implantation significantly accelerated wound healing and increased cellularity and re-epithelialization. Transplanted AMEs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, suggesting direct benefits for cutaneous closure. Taken together, these data indicate that AMEs possess therapeutic capability for wound healing through the secretion of epithelialization growth factors and enhanced engraftment properties. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26174407

  1. Altered expression of apoptotic genes in response to OCT4B1 suppression in human tumor cell lines.

    PubMed

    Mirzaei, Mohammad Reza; Najafi, Ali; Arababadi, Mohammad Kazemi; Asadi, Malek Hosein; Mowla, Seyed Javad

    2014-10-01

    OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1. PMID:25008565

  2. Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

    PubMed

    de Castro, M A G; Bunt, G; Wouters, F S

    2016-01-01

    The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B's proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis. PMID:27551506

  3. Apoptotic effects of non-edible parts of Punica granatum on human multiple myeloma cells.

    PubMed

    Kiraz, Yağmur; Neergheen-Bhujun, Vidushi S; Rummun, Nawraj; Baran, Yusuf

    2016-02-01

    Multiple myeloma is of great concern since existing therapies are unable to cure this clinical condition. Alternative therapeutic approaches are mandatory, and the use of plant extracts is considered interesting. Punica granatum and its derived products were suggested as potential anticancer agents due to the presence of bioactive compounds. Thus, polypenolic-rich extracts of the non-edible parts of P. granatum were investigated for their antiproliferative and apoptotic effects on U266 multiple myeloma cells. We demonstrated that there were dose-dependent decreases in the proliferation of U266 cells in response to P. granatum extracts. Also, exposure to the extracts triggered apoptosis with significant increases in loss of mitochondrial membrane potential in U266 cells exposed to the leaves and stem extracts, while the flower extract resulted in slight increases in loss of MMP. These results were confirmed by Annexin-V analysis. These results documented the cytotoxic and apoptotic effects of P. granatum extracts on human U266 multiple myeloma cells via disruption of mitochondrial membrane potential and increasing cell cycle arrest. The data suggest that the extracts can be envisaged in cancer chemoprevention and call for further exploration into the potential application of these plant parts. PMID:26318303

  4. Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes

    PubMed Central

    de Castro, MAG; Bunt, G; Wouters, FS

    2016-01-01

    The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B’s proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis. PMID:27551506

  5. [Effect of lidamycin on mitochondria initiated apoptotic pathway in human cancer cells].

    PubMed

    Qiu, Qiang; Wang, Zhen; Jiang, Jian-ming; Li, Dian-dong

    2007-02-01

    Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities. PMID:17518039

  6. Capsaicinoids Cause Inflammation and Epithelial Cell Death through Activation of Vanilloid Receptors

    PubMed Central

    Reilly, Christopher A.; Taylor, Jack L.; Lanza, Diane L.; Carr, Brian A.; Crouch, Dennis J.; Yost, Garold S.

    2008-01-01

    Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results

  7. Capsaicinoids cause inflammation and epithelial cell death through activation of vanilloid receptors.

    PubMed

    Reilly, Christopher A; Taylor, Jack L; Lanza, Diane L; Carr, Brian A; Crouch, Dennis J; Yost, Garold S

    2003-05-01

    Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results

  8. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

    SciTech Connect

    Katano, Takahito; Ootani, Akifumi; Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi; Toda, Shuji; Joh, Takashi

    2013-03-22

    Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.

  9. Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells

    PubMed Central

    Lee, Jong Soo; Lee, Seung Uk; Che, Cheng-Ye; Lee, Ji-Eun

    2015-01-01

    AIM To investigate the cytotoxic effect on human corneal epithelial cells (HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose (CMC) and hyaluronic acid (HA). METHODS HCECs were exposed to 0.5% CMC (Refresh plus®, Allergan, Irvine, California, USA) and 0.1% and 0.3% HA (Kynex®, Alcon, Seoul, Korea, and Hyalein mini®, Santen, Osaka, Japan) for the period of 30min, and 4, 12, and 24h. Methyl thiazolyl tetrazolium (MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase (LDH) leakage assay to assess the cytotoxicity. Apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24h after confluent HCECs were scratch wounded. RESULTS The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells were demonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC. CONCLUSION CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds. PMID:25938030

  10. Apoptotic-like programed cell death in fungi: the benefits in filamentous species

    PubMed Central

    Shlezinger, Neta; Goldfinger, Nir; Sharon, Amir

    2012-01-01

    Studies conducted in the early 1990s showed for the first time that Saccharomyces cerevisiae can undergo cell death with hallmarks of animal apoptosis. These findings came as a surprise, since suicide machinery was unexpected in unicellular organisms. Today, apoptosis in yeast is well-documented. Apoptotic death of yeast cells has been described under various conditions and S. cerevisiae homologs of human apoptotic genes have been identified and characterized. These studies also revealed fundamental differences between yeast and animal apoptosis; in S. cerevisiae apoptosis is mainly associated with aging and stress adaptation, unlike animal apoptosis, which is essential for proper development. Further, many apoptosis regulatory genes are either missing, or highly divergent in S. cerevisiae. Therefore, in this review we will use the term apoptosis-like programed cell death (PCD) instead of apoptosis. Despite these significant differences, S. cerevisiae has been instrumental in promoting the study of heterologous apoptotic proteins, particularly from human. Work in fungi other than S. cerevisiae revealed differences in the manifestation of PCD in single cell (yeasts) and multicellular (filamentous) species. Such differences may reflect the higher complexity level of filamentous species, and hence the involvement of PCD in a wider range of processes and life styles. It is also expected that differences might be found in the apoptosis apparatus of yeast and filamentous species. In this review we focus on aspects of PCD that are unique or can be better studied in filamentous species. We will highlight the similarities and differences of the PCD machinery between yeast and filamentous species and show the value of using S. cerevisiae along with filamentous species to study apoptosis. PMID:22891165

  11. The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells.

    PubMed

    Liang, Wei-Zhe; Chou, Chiang-Ting; Hsu, Shu-Shong; Liao, Wei-Chuan; Shieh, Pochuen; Kuo, Daih-Huang; Tseng, Hui-Wen; Kuo, Chun-Chi; Jan, Chung-Ren

    2015-01-01

    Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca(2+) levels ([Ca(2+)]i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca(2+)]i rises which were reduced by removing extracellular Ca(2+). Eugenol-induced [Ca(2+)]i rises were not altered by store-operated Ca(2+) channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca(2+)]i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca(2+)]i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca(2+)]i rises by inducing PLC-dependent release of Ca(2+) from the endoplasmic reticulum and caused Ca(2+) influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway. PMID:25455450

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

    PubMed

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

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway. PMID:26739898

  13. Different apoptotic effects of saxifragifolin C in human breast cancer cells.

    PubMed

    Kim, Kyung-Ho; Kim, Ji-Yun; Kwak, Jong-Hwan; Kim, Byung Oh; Pyo, Suhkneung

    2016-04-01

    Breast cancer is currently the most common form of cancer affecting women. Recent studies have reported that triterpenoid saponins isolated from Androsace umbellata exhibit anti-proliferative effects in several types of cancer cells. However, the cytotoxic effect of saxifragifolin C (Saxi C) on breast cancer cells remains unclear. The purpose of this study is to evaluate the in vitro anti-tumor activity of Saxi C in human breast cancer cells. Our data indicated that MDA-MB-231 cells were more sensitive than MCF-7 cells to Saxi C treatment. In addition, Saxi C inhibited cell survival through the induction of reactive oxygen species and the caspase-dependent pathway in the MDA-MB-231 cells, whereas MCF-7 cells treated with Saxi C underwent the apoptotic cell death in a caspase-independent manner. Although Saxi C treatment resulted in the induction of activation of MAPKs in both types of human breast cancer cells, p38 MAPK and JNK, but not ERK1/2, appeared to be involved in Saxi C-induced apoptosis. Moreover, ERα-overexpressing MDA-MB-231 cells remained alive, whereas the survival of shERα-transfected MCF-7 cells decreased. Taken together, Saxi C induced apoptosis in MCF-7 cells and MDA-MB-231 cells via different regulatory mechanisms, and ERα status might be essential for regulating Saxi C-induced apoptosis in breast cancer cells. Thus, Saxi C is a potential chemotherapeutic agent in breast cancer. PMID:26965415

  14. Salivary epithelial cells: an unassuming target site for gene therapeutics

    PubMed Central

    Perez, Paola; Rowzee, Anne M.; Zheng, Changyu; Adriaansen, Janik; Baum, Bruce J.

    2010-01-01

    Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer. PMID:20219693

  15. Phloroglucinol induces apoptosis via apoptotic signaling pathways in HT-29 colon cancer cells

    PubMed Central

    KANG, MI-HYE; KIM, IN-HYE; NAM, TAEK-JEO NG

    2014-01-01

    Phloroglucinol is a polyphenolic compound that is used to treat and prevent several human diseases, as it exerts beneficial biological activities, including anti-oxidant, anti-inflammatory and anticancer properties. The aim of the present study was to investigate the effects of phloroglucinol on apoptotic signaling pathways in HT-29 colon cancer cells. The results indicated that phloroglucinol suppressed cell viability and induced apoptosis in HT-29 cells in a concentration-dependent manner. Phloroglucinol treatment of HT-29 cells resulted in characteristic apoptosis-related changes: altered Bcl-2 family proteins, cytochrome c release, and activation of caspase-3 and caspase-8. This study also showed that proteins involved in apoptosis were stimulated by treatment with phloroglucinol. These findings demonstrated that phloroglucinol exerts anticancer activity in HT-29 colon cancer cells through induction of apoptosis. PMID:25070748

  16. Establishment and Characterization of Immortalized Human Amniotic Epithelial Cells

    PubMed Central

    Zhou, Kaixuan; Koike, Chika; Yoshida, Toshiko; Okabe, Motonori; Fathy, Moustafa; Kyo, Satoru; Kiyono, Tohru; Saito, Shigeru

    2013-01-01

    Abstract Human amniotic epithelial cells (HAEs) have a low immunogenic profile and possess potent immunosuppressive properties. HAEs also have several characteristics similar to stem cells, and they are discarded after parturition. Thus, they could potentially be used in cell therapy with fewer ethical problems. HAEs have a short life, so our aim is to establish and characterize immortalized human amniotic epithelial cells (iHAEs). HAEs were introduced with viral oncogenes E6/E7 and with human telomerase reverse transcriptase (hTERT) to create iHAEs. These iHAEs have proliferated around 200 population doublings (PDs) for at least 12 months. High expression of stem cell markers (Oct 3/4, Nanog, Sox2, Klf4) and epithelial markers (CK5, CK18) were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). These iHAEs were expanded in ultra-low-attachment dishes to form spheroids similarly to epithelial stem/precursor cells. High expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers was detected by flow cytometry. The iHAEs showed adipogenic, osteogenic, neuronal, and cardiac differentiation abilities. In conclusion, the immortalization of HAEs with the characteristics of stem cells has been established, allowing these iHAEs to become useful for cell therapy and regenerative medicine. PMID:23298399

  17. Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptosis in vitro.

    PubMed

    Firth, James D; Putnins, Edward E

    2004-01-01

    The ability of keratinocyte growth factor 1 to modulate apoptosis in the absence of proliferation was studied in vitro. A HaCaT scrape wound model was developed in which dense monolayers prior to wounding were cultured to quiescence in defined media with hydroxyurea at concentrations that blocked proliferation without loss of cell viability. Scrape wounding was then found to induce apoptosis, originating at the wound edge, but subsequently radiating away over a 24 h period to encompass areas not originally damaged. Keratinocyte growth factor 1 inhibited this radial progression of apoptosis in a concentration-dependent manner up to 20 ng per mL with induced migration present at the wound edge. The extent of this rescue was modulated by the concentration of Ca2+ prior to wounding. In control wound cultures apoptotic bodies were found in cells adjacent to the wound interface but were greatly reduced in keratinocyte-growth-factor-1-treated groups. Keratinocyte growth factor 1 receptor expression was significantly induced within two to three cell widths of the scraped wound edge, at levels far exceeding those found at the leading edge of a nonwounded epithelial sheet. Tumor necrosis factor alpha (1-5 ng per mL) or Escherichia coli lipopolysaccharide (10-50 ng per mL) exacerbated scrape-induced early apoptosis (1-4 h), but was largely ameliorated by coculture with keratinocyte growth factor 1. Keratinocyte growth factor 1 protection was associated with a reduction in both caspase-3 activation and cytokeratin-19 loss. Protected wound edges were also associated with the maintenance of e-cadherin expression and induction of beta1 integrin and actin stress fiber organization. These results suggest that keratinocyte growth factor 1 may play a role in limiting mechanically induced apoptotic processes at the epithelial wound edge in a manner that is distinct from its proliferative function. PMID:14962112

  18. Decline in NAD(P)H autofluorescence precedes apoptotic cell death from chemotherapy

    NASA Astrophysics Data System (ADS)

    Toms, Steven A.; Muhammad, Osman; Jackson, Heather; Lin, Wei-Chiang

    2005-11-01

    OBJECTIVE: Optical spectroscopic tools exist that allow open surgical and minimally invasive assays of intrinsic tissue optics. Optical detection of cellular and tissue viability may offer a minimally invasive way to assess tumor responsiveness to chemotherapies. We report on an optical spectroscopic change that precedes apoptotic cell death and appears related to NAD(P)H autofluorescence. METHODS: The cell lines SW 480 and U87-MG were grown in culture and treated with cisplatin 100 μg/ml and tamoxifen 10 μM, respectively. Fluorescence spectroscopy at 355 nm excitation and 460 nm emission were collected. MTS assays were used to determine cell viability. Cell lysates were analyzed for NAD(P)H concentrations by mass spectroscopy. RESULTS: Autoflourescence at 355 nm excitation and 460 nm emission declines markedly despite normalization for cell number and total protein concentration after treatment with tamoxifen or cisplatin. The autofluorescence drop precedes the loss of cell viability as measured by MTS assay. For example, the relative viability of the U87-MG cell treated with tamoxifen at hours 0, 8, 12 and 24 of treatment was 100 +/- 6, 85 +/- 6, 53 +/- 9 and 0 +/- 3. The relative fluorescence at the same time points were 100 +/- 2, 57 +/- 6, 47 +/- 3, and 0 +/- 1. TUNNEL assays confirm that cell death is via apoptosis. The key cellular fluorophore at these wavelengths is NAD(P)H. Mass spectroscopic analysis of cell lysates at these time points reveals a drop in NAD(P)H concentrations that is parallel to the loss of fluorescence signal. CONCLUSIONS: NAD(P)H autofluoresence decline precedes apoptotic cell death. This may allow the design of minimally invasive spectroscopic tools to monitor chemotherapeutic response.

  19. Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells

    PubMed Central

    Bayles, Kenneth W.; Wesson, Carla A.; Liou, Linda E.; Fox, Lawrence K.; Bohach, Gregory A.; Trumble, W. R.

    1998-01-01

    We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes. PMID:9423876

  20. Freezing and post-thaw apoptotic behaviour of cells in the presence of palmitoyl nanogold particles

    NASA Astrophysics Data System (ADS)

    Thirumala, Sreedhar; Forman, Julianne M.; Monroe, W. Todd; Devireddy, Ram V.

    2007-05-01

    The aim of this study was to evaluate the freezing response of HeLa and Jurkat cells in the presence of commercially available nanoparticles, NPs (Palmitoyl Nanogold®, Nanoprobes). The cells were incubated with NPs for either 5 min or 3 h, and a calorimeter technique was then used to generate the volumetric shrinkage response during freezing at 20 °C min-1. Concomitantly, we also examined the effect of a commonly used cryoprotectant, dimethylsulfoxide, DMSO (10% v/v ratio) on the freezing response of HeLa and Jurkat cells. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the reference hydraulic conductivity, Lpg, (μm/min-atm) and activation energy, ELp, (kcal mol-1) were obtained. For HeLa cells, the values of Lpg ranged from 0.08 to 0.23 µm/min-atm, while ELp ranged from 10.9 to 37.4 kcal mol-1. For Jurkat cells these parameter values ranged from 0.05 to 0.16 µm/min-atm and 9.5 to 35.9 kcal mol-1. A generic optimal cooling rate equation was then used to predict the optimal rates of freezing HeLa and Jurkat cells in the presence and absence of DMSO and NPs. The post-thaw viability and apoptotic response of HeLa and Jurkat cells was further investigated by cooling cells at three rates in the presence and absence of DMSO and NPs using a commercially available controlled rate freezer. Jurkat cells treated in this manner demonstrated an increase in their adhesive properties after 18 h incubation and adhered strongly to the bottom of the culture plate. This observation prevented further analysis of Jurkat apoptotic and necrotic post-thaw responses. There was no significant effect of NPs or DMSO alone on HeLa cell viability prior to freezing. The post-thaw results from HeLa cells show that the NPs increased the measured post-freeze apoptotic response when cooled at 1 °C min-1, suggesting a possible therapeutic use of NPs in cryodestructive procedures.

  1. Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation[S

    PubMed Central

    Haka, Abigail S.; Barbosa-Lorenzi, Valéria C.; Lee, Hyuek Jong; Falcone, Domenick J.; Hudis, Clifford A.; Dannenberg, Andrew J.

    2016-01-01

    Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. PMID:27044658

  2. Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration*

    PubMed Central

    Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.

    2015-01-01

    Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently. PMID:25713081

  3. Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation.

    PubMed

    Haka, Abigail S; Barbosa-Lorenzi, Valéria C; Lee, Hyuek Jong; Falcone, Domenick J; Hudis, Clifford A; Dannenberg, Andrew J; Maxfield, Frederick R

    2016-06-01

    Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. PMID:27044658

  4. A novel pathway for phagosome maturation during engulfment of apoptotic cells

    PubMed Central

    Kinchen, Jason M.; Doukoumetzidis, Kimon; Almendinger, Johann; Stergiou, Lilli; Tosello-Trampont, Annie; Sifri, Costi D.; Hengartner, Michael O.; Ravichandran, Kodi S.

    2010-01-01

    The efficient removal of apoptotic cells is critical for the physiological well-being of the organism1â4; defects in corpse removal have been linked to autoimmune disease4, 5. While several players regulating the early steps of corpse recognition and internalization have been characterized6, the molecules and mechanisms relevant to the subsequent processing of the internalized corpses are poorly understood. Here, we identify a novel pathway for the processing of internalized apoptotic cells in C. elegans and in mammals. First, we show that RAB-5 and RAB-7 are sequentially recruited to phagosomes containing apoptotic corpses as they mature within phagocytes, and that both proteins are required for efficient corpse clearance. We then used targeted genetic screens to identify players regulating the recruitment and/or retention of Rab5 and Rab7 to phagosomes. Seven members of the HOPS complex (a Rab7 activator/effector complex) were required for Rab7 localization or retention on phagosomes. In an effort to identify factors that regulate Rab5 recruitment, we undertook an unbiased reverse genetic screen and identified 61 genes potentially required for corpse removal. In-depth analysis of two candidate genes, vps-34 and dyn-1/dynamin, showed accumulation of internalized, but undegraded corpses within abnormal phagosomes that are defective in RAB-5 recruitment. Using a series of genetic and biochemical experiments in worms and mammalian cells, we ordered these proteins in a pathway, with DYN-1 functioning upstream of VPS-34, in the recruitment/retention of Rab5 to the nascent phagosome. Further, we identified a novel biochemical complex containing Vps34, dynamin and Rab5GDP, providing a mechanism for Rab5 recruitment to the nascent phagosome. PMID:18425118

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

    EPA Science Inventory

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

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

    PubMed Central

    St Johnston, Daniel

    2016-01-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium1,2. Here we test this assumption in three types of Drosophila epithelia; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside of the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells appears to be driven by lateral adhesion, which pulls cells born outside the epithelia layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  7. Epithelial stem cells and implications for wound repair.

    PubMed

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

    2012-12-01

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

  8. Epithelial Stem Cells and Implications for Wound Repair

    PubMed Central

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

    2012-01-01

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

  9. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

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

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

    PubMed

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

    2007-03-01

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

  11. The syncytial nature of epithelial cells in the thymic cortex.

    PubMed Central

    Kendall, M D

    1986-01-01

    The epithelial cells of the cortex of human and rodent thymus glands were examined by light and electron microscopy, and the intracellular membrane potentials measured from the subcapsular, cortical and medullary regions. In the human thymus cortex, there is a highly correlated age-independent relationship (r = 0.78) between the distance in micron from one adjacent Type 2/3 epithelial nucleus to another, and the number of thymocytes between them. In rodent glands that had undergone some degree of involution due to hypoxia simulating an altitude of 17 000 feet or following the injection of phenylhydrazine, Type 2/3 epithelial cells were often found to be bi- or multinucleated. Electrophysiological studies of 10 mouse thymus lobes using 0.2 micron tipped electrodes showed that there were highly significant differences (P less than 0.0001) between the intracellular membrane potentials of the subcapsular zone, the cortex and the medulla. When dyes were injected intracellularly (through 0.5 micron tipped electrodes) into individual epithelial cells, methylene blue remained within the cytoplasm, but procion yellow passed in 30 minutes into the nuclei of all the epithelial cells of the cortex but not those of the subcapsular zone, nor the medulla. This indicates that the cortex must be a functional syncytium and it differs in this respect from the rest of the gland. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 PMID:3319999

  12. Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

    PubMed Central

    Maruthamuthu, Venkat; Gardel, Margaret L.

    2014-01-01

    Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering. PMID:25099795

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

    PubMed Central

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

    2015-01-01

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

  14. Induction of apoptotic cell death leads to the development of bacterial rot caused by Pseudomonas cichorii.

    PubMed

    Kiba, Akinori; Sangawa, Yasutaka; Ohnishi, Kouhei; Yao, Nan; Park, Pyoyun; Nakayashiki, Hitoshi; Tosa, Yukio; Mayama, Shigeyuki; Hikichi, Yasufumi

    2006-02-01

    Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P. cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii. PMID:16529373

  15. Increase in proliferation and apoptosis of gastric epithelial cells early in the natural history of Helicobacter pylori infection.

    PubMed Central

    Jones, N. L.; Shannon, P. T.; Cutz, E.; Yeger, H.; Sherman, P. M.

    1997-01-01

    Childhood acquisition of Helicobacter pylori is a critical risk factor for gastric cancer. Since tumorigenesis involves deregulation of proliferation and apoptosis, we examined gastric epithelial cell proliferation and apoptosis in H. pylori-infected children. Apoptosis and proliferation of gastric antral epithelial cells in biopsy specimens from patients with H. pylori-induced gastritis, secondary gastritis, and noninflamed controls were compared. p53 protein expression was examined immunohistochemically. Apoptotic cells were identified in the surface epithelium in each group. The apoptotic index was higher in specimens from patients with H. pylori gastritis (120 +/- 10) than secondary gastritis (50 +/- 10) and noninflamed controls (40 +/- 10, analysis of variance P < 0.005). Apoptosis decreased following H. pylori eradication and resolution of gastritis (P < 0.02). An expanded proliferative compartment was identified in H. pylori-induced gastritis (32.4 +/- 3.5; proliferative labeling index +/- SE) compared with secondary gastritis (18.9 +/- 2.8) and noninflamed controls (13.7 +/- 3.1, analysis of variance P < 0.01). The accelerated cell turnover was associated with p53 overexpression (analysis of variance P < 0.005). Accumulation of p53 was not associated with expression of the cyclin-dependent kinase inhibitor p21. The occurrence of altered cell turnover early in the natural history of chronic infection provides an explanation for the increased risk of gastric cancer development associated with childhood acquisition of infection. Images Figure 1 Figure 4 Figure 6 Figure 8 PMID:9403720

  16. [Epithelial cell in intestinal homeostasis and inflammatory bowel diseases].

    PubMed

    Zouiten-Mekki, Lilia; Serghini, Meriem; Fekih, Monia; Kallel, Lamia; Matri, Samira; Ben Mustapha, Nadia; Boubaker, Jalel; Filali, Azza

    2013-12-01

    Crohn's disease (CD) and ulcerative colitis (UC) are the principal inflammatory bowel diseases (IBD) which physiopathology is currently poorly elucidated. During these diseases, the participation of the epithelial cell in the installation and the perpetuation of the intestinal inflammation is now clearly implicated. In fact, the intestinal epithelium located at the interface between the internal environment and the intestinal luminal, is key to the homeostatic regulation of the intestinal barrier. This barrier can schematically be regarded as being three barriers in one: a physical, chemical and immune barrier. The barrier function of epithelial cell can be altered by various mechanisms as occurs in IBD. The goal of this article is to review the literature on the role of the epithelial cell in intestinal homeostasis and its implication in the IBD. PMID:24356146

  17. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

    PubMed

    Verghese, Shilpi; Su, Tin Tin

    2016-09-01

    Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue. PMID:27584613

  18. Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase 3 activation in HL-60 cells.

    PubMed

    Huang, R F; Huang, S M; Lin, B S; Wei, J S; Liu, T Z

    2001-05-11

    The cytotoxicity of homocysteine derivatives on chromosomal damage in somatic cells is not well established. The present study used reactive homocysteine derivative of homocysteine thiolactone (Hcy) to investigate its causal effect on apoptotic DNA injury in human promyeloid HL-60 cells. Our results demonstrated that Hcy induced cell death and features of apoptosis including increased phosphotidylserine exposure on the membrane surface, increased apoptotic cells with hypoploid DNA contents, and internucleosomal DNA fragmentation, all of which occurred in a time- and concentration-dependent manner. Hcy treatment also significantly increased intracellular reactive oxygen species H2O2, which coincided with the elimination of caspase 3 proenzyme levels and increased caspase 3 activity at the time of the appearance of apoptotic DNA fragmentation. Preincubation of Hcy-treated HL-60 cells with catalase completely scavenged intracellular H2O2, thus inhibiting caspase 3 activity and protecting cells from apoptotic DNA damage. In contrast, superoxide dismutase failed to inhibit Hcy-induced DNA damage. Taken together, these results demonstrate that Hcy exerted its genotoxic effects on HL-60 cells through an apoptotic pathway, which is mediated by the activation of caspase 3 activity induced by an increase in intracellular hydrogen peroxide. PMID:11432446

  19. Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice.

    PubMed

    Bhowmick, Debajit; Bhar, Kaushik; Mallick, Sanjaya K; Das, Subhadip; Chatterjee, Nabanita; Sarkar, Tuhin Subhra; Chakrabarti, Rajarshi; Das Saha, Krishna; Siddhanta, Anirban

    2016-01-01

    Melanoma is one of the most aggressive forms of cancer, usually resistant to standard chemotherapeutics. Despite a huge number of clinical trials, any success to find a chemotherapeutic agent that can effectively destroy melanoma is yet to be achieved. Para-phenylenediamine (p-PD) in the hair dyes is reported to purely serve as an external dyeing agent. Very little is known about whether p-PD has any effect on the melanin producing cells. We have demonstrated p-PD mediated apoptotic death of both human and mouse melanoma cells in vitro. Mouse melanoma tumour growth was also arrested by the apoptotic activity of intraperitoneal administration of p-PD with almost no side effects. This apoptosis is shown to occur primarily via loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS), and caspase 8 activation. p-PD mediated apoptosis was also confirmed by the increase in sub-G0/G1 cell number. Thus, our experimental observation suggests that p-PD can be a potential less expensive candidate to be developed as a chemotherapeutic agent for melanoma. PMID:27293892

  20. Agmatine Attenuates Brain Edema and Apoptotic Cell Death after Traumatic Brain Injury.

    PubMed

    Kim, Jae Young; Lee, Yong Woo; Kim, Jae Hwan; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2015-07-01

    Traumatic brain injury (TBI) is associated with poor neurological outcome, including necrosis and brain edema. In this study, we investigated whether agmatine treatment reduces edema and apoptotic cell death after TBI. TBI was produced by cold injury to the cerebral primary motor cortex of rats. Agmatine was administered 30 min after injury and once daily until the end of the experiment. Animals were sacrificed for analysis at 1, 2, or 7 days after the injury. Various neurological analyses were performed to investigate disruption of the blood-brain barrier (BBB) and neurological dysfunction after TBI. To examine the extent of brain edema after TBI, the expression of aquaporins (AQPs), phosphorylation of mitogen-activated protein kinases (MAPKs), and nuclear translocation of nuclear factor-κB (NF-κB) were investigated. Our findings demonstrated that agmatine treatment significantly reduces brain edema after TBI by suppressing the expression of AQP1, 4, and 9. In addition, agmatine treatment significantly reduced apoptotic cell death by suppressing the phosphorylation of MAPKs and by increasing the nuclear translocation of NF-κB after TBI. These results suggest that agmatine treatment may have therapeutic potential for brain edema and neural cell death in various central nervous system diseases. PMID:26130959

  1. Role of mitochondria in apoptotic and necroptotic cell death in the developing brain

    PubMed Central

    Thornton, Claire; Hagberg, Henrik

    2015-01-01

    Hypoxic–ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short- and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia–ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia–ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome (composed of RIP1, RIP3 and MLKL), both of which converge at the mitochondria. PMID:25661091

  2. Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice

    PubMed Central

    Bhowmick, Debajit; Bhar, Kaushik; Mallick, Sanjaya K.; Das, Subhadip; Chatterjee, Nabanita; Sarkar, Tuhin Subhra; Chakrabarti, Rajarshi; Das Saha, Krishna; Siddhanta, Anirban

    2016-01-01

    Melanoma is one of the most aggressive forms of cancer, usually resistant to standard chemotherapeutics. Despite a huge number of clinical trials, any success to find a chemotherapeutic agent that can effectively destroy melanoma is yet to be achieved. Para-phenylenediamine (p-PD) in the hair dyes is reported to purely serve as an external dyeing agent. Very little is known about whether p-PD has any effect on the melanin producing cells. We have demonstrated p-PD mediated apoptotic death of both human and mouse melanoma cells in vitro. Mouse melanoma tumour growth was also arrested by the apoptotic activity of intraperitoneal administration of p-PD with almost no side effects. This apoptosis is shown to occur primarily via loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS), and caspase 8 activation. p-PD mediated apoptosis was also confirmed by the increase in sub-G0/G1 cell number. Thus, our experimental observation suggests that p-PD can be a potential less expensive candidate to be developed as a chemotherapeutic agent for melanoma. PMID:27293892

  3. Chelidonine induces mitotic slippage and apoptotic-like death in SGC-7901 human gastric carcinoma cells.

    PubMed

    Qu, Zhongyuan; Zou, Xiang; Zhang, Xiujuan; Sheng, Jiejing; Wang, Yumeng; Wang, Jiaqi; Wang, Chao; Ji, Yubin

    2016-02-01

    The aim of the present study was to investigate the effect of chelidonine on mitotic slippage and apoptotic-like death in SGC-7901 human gastric cancer cells. The MTT assay was performed to detect the antiproliferative effect of chelidonine. Following treatment with chelidonine (10 µmol/l), the ultrastructure changes in SGC-7901, MCF-7 and HepG2 cells were observed by transmission electron microscopy. The effects of chelidonine on G2/M phase arrest and apoptosis of SGC-7901 cells were determined by flow cytometry. Indirect immunofluorescence assay and laser scanning confocal microscopy (LSCM) were used to detect the phosphorylation level of histone H3 (Ser10) and microtubule formation was detected using LSCM following immunofluorescent labeling. Subsequent to treatment with chelidonine (10 µmol/l), expression levels of mitotic slippage-associated proteins, including BUB1 mitotic checkpoint serine/threonine kinase B (BubR1), cyclin-dependent kinase 1 (Cdk1) and cyclin B1, and apoptosis-associated protein, caspase-3 were examined by western blotting at 24, 48 and 72 h. The half maximal inhibitory concentration of chelidonine was 23.13 µmol/l over 48 h and chelidonine induced G2/M phase arrest of cells. The phosphorylation of histone H3 at Ser10 was significantly increased following treatment with chelidonine for 24 h, indicating that chelidonine arrested the SGC-7901 cells in the M phase. Chelidonine inhibited microtubule polymerization, destroyed microtubule structures and induced cell cycle arrest in the M phase. Giant cells were observed with multiple micronuclei of varying sizes, which indicated that following a prolonged arrest in the M phase, the cells underwent mitotic catastrophe. Western blotting demonstrated that the protein expression levels of BubR1, cyclin B1 and Cdk1 decreased significantly between 48 and 72 h. Low expression levels of BubR1 and inactivation of the cyclin B1-Cdk1 complex results in the cells being arrested at mitosis and leads to

  4. Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

    PubMed Central

    Wang, X; Gerdes, H-H

    2015-01-01

    Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context. PMID:25571977

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

    PubMed Central

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

    2014-01-01

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

  6. Apoptotic pathways as a therapeutic target for colorectal cancer treatment

    PubMed Central

    Abraha, Aman M; Ketema, Ezra B

    2016-01-01

    Colorectal cancer is the second leading cause of death from cancer among adults. The disease begins as a benign adenomatous polyp, which develops into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer. Appropriate apoptotic signaling is fundamentally important to preserve a healthy balance between cell death and cell survival and in maintaining genome integrity. Evasion of apoptotic pathway has been established as a prominent hallmark of several cancers. During colorectal cancer development, the balance between the rates of cell growth and apoptosis that maintains intestinal epithelial cell homeostasis gets progressively disturbed. Evidences are increasingly available to support the hypothesis that failure of apoptosis may be an important factor in the evolution of colorectal cancer and its poor response to chemotherapy and radiation. The other reason for targeting apoptotic pathway in the treatment of cancer is based on the observation that this process is deregulated in cancer cells but not in normal cells. As a result, colorectal cancer therapies designed to stimulate apoptosis in target cells would play a critical role in controlling its development and progression. A better understanding of the apoptotic signaling pathways, and the mechanisms by which cancer cells evade apoptotic death might lead to effective therapeutic strategies to inhibit cancer cell proliferation with minimal toxicity and high responses to chemotherapy. In this review, we analyzed the current understanding and future promises of apoptotic pathways as a therapeutic target in colorectal cancer treatment. PMID:27574550

  7. Apoptotic pathways as a therapeutic target for colorectal cancer treatment.

    PubMed

    Abraha, Aman M; Ketema, Ezra B

    2016-08-15

    Colorectal cancer is the second leading cause of death from cancer among adults. The disease begins as a benign adenomatous polyp, which develops into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer. Appropriate apoptotic signaling is fundamentally important to preserve a healthy balance between cell death and cell survival and in maintaining genome integrity. Evasion of apoptotic pathway has been established as a prominent hallmark of several cancers. During colorectal cancer development, the balance between the rates of cell growth and apoptosis that maintains intestinal epithelial cell homeostasis gets progressively disturbed. Evidences are increasingly available to support the hypothesis that failure of apoptosis may be an important factor in the evolution of colorectal cancer and its poor response to chemotherapy and radiation. The other reason for targeting apoptotic pathway in the treatment of cancer is based on the observation that this process is deregulated in cancer cells but not in normal cells. As a result, colorectal cancer therapies designed to stimulate apoptosis in target cells would play a critical role in controlling its development and progression. A better understanding of the apoptotic signaling pathways, and the mechanisms by which cancer cells evade apoptotic death might lead to effective therapeutic strategies to inhibit cancer cell proliferation with minimal toxicity and high responses to chemotherapy. In this review, we analyzed the current understanding and future promises of apoptotic pathways as a therapeutic target in colorectal cancer treatment. PMID:27574550

  8. Epithelial cell cultures from normal and cancerous human tissues.

    PubMed

    Owens, R B; Smith, H S; Nelson-Rees, W A; Springer, E L

    1976-04-01

    Thirty epithelial cell strains were isolated from human carcinomas and normal epithelial tissues by collagenase digestion and selective removal of fibroblasts with trypsin-Versene. Most strains were obtained from metastatic carcinomas or epithelia of the urinary and intestinal tracts. The success rate for growth of both neoplastic and normal tissues (excluding skin) was 38%. Six of these strains showed gross morphologic and chromosome changes typical of malignant cells. Nine resembled normal epithelium. The other 15 exhibited some degree of morphologic change from normal. PMID:176412

  9. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    PubMed

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. PMID:27189858

  10. Comparison of the Kinetics of Maturation of Phagosomes Containing Apoptotic Cells and IgG-Opsonized Particles

    PubMed Central

    Viegas, Michelle S.; Estronca, Luís M. B. B.; Vieira, Otília V.

    2012-01-01

    Defective clearance of apoptotic cells has emerged as an important contributing factor to the pathogenesis of many diseases. Although many efforts have been made to understand the machinery involved in the recognition between phagocytes and potential targets, little is known about the intracellular transport of phagosomes containing apoptotic cells within mammalian cells. We have, therefore, performed a detailed study on the maturation of phagosomes containing apoptotic cells in a non-professional phagocytic cell line. This process was compared with the maturation of IgG-opsonized particles, which are internalized via the Fcγ-receptor (Fcγ-R), one of the best characterized phagocytic receptor, in the same cell line stably expressing the Fcγ-RIIA. By comparing markers from different stages of phagosome maturation, we have found that phagosomes carrying apoptotic particles reach the lysosomes with a delay compared to those containing IgG-opsonized particles. Enrichment of the apoptotic particles in phosphatidylserine (PS) neither changed the kinetics of their engulfment nor the maturation process of the phagosome. PMID:23119002

  11. Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro

    PubMed Central

    Wang, Jing; Yang, Yangfan; Xu, Jiangang; Lin, Xianchai; Wu, Kaili

    2013-01-01

    Purpose To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. Methods Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFβS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFβS, FN, and Snail1 were analyzed using reverse transcriptase–polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. Results PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFβ1, TGFβ2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFβ1, and TGFβ2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. Conclusions PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity. PMID:24415895

  12. Expression of the FGFR2 mesenchymal splicing variant in epithelial cells drives epithelial-mesenchymal transition

    PubMed Central

    Ranieri, Danilo; Rosato, Benedetta; Nanni, Monica; Magenta, Alessandra

    2016-01-01

    The FGFRs are receptor tyrosine kinases expressed by tissue-specific alternative splicing in epithelial IIIb or mesenchymal IIIc isoforms. Deregulation of FGF/FGFR signaling unbalances the epithelial-stromal homeostasis and may lead to cancer development. In the epithelial-context, while FGFR2b/KGFR acts as tumor suppressor, FGFR2c appears to play an oncogenic role. Based on our recent observation that the switching of FGFR2b versus FGFR2c induces EMT, here we investigated the biological outcome of the ectopic expression of FGFR2c in normal human keratinocytes. Morphological analysis showed that, differently from FGFR2b overexpression, the forced expression and activation of FGFR2c drive the epithelial cells to acquire a mesenchymal-like shape and actin reorganization. Moreover, the appearance of invasiveness and anchorage-independent growth ability in FGFR2c transfected keratinocytes was consistent with the potential tumorigenic role proposed for this receptor variant. Biochemical and molecular approaches revealed that the observed phenotypic changes were accompanied by modulation of EMT biomarkers and indicated the involvement of EMT transcription factors and miRs. Finally, the analysis of the expression pattern of discriminating markers strongly suggested that activation of FGFR2c triggers a process corresponding to the initiation of the pathological type III EMT, but not to the more physiological type II EMT occurring during FGFR2b-mediated wound healing. PMID:26713601

  13. Counteraction of Apoptotic and Inflammatory Effects of Adriamycin in the Liver Cell Culture by Clinopitolite.

    PubMed

    Yapislar, Hande; Taskin, Eylem; Ozdas, Sule; Akin, Demet; Sonmez, Emine

    2016-04-01

    Growing evidence has been reported on adriamycin (ADR) hepatotoxicity in literature. Hepatotoxicity caused by the use of drugs has a serious undesirable effect in the cure of cancer patients that needs to be eliminated. The exact mechanism of ADR on non-cancerous tissue still remains to be a mystery. The zeolite (clinoptilolite) minerals form a complex group of aluminosilicates that often occur as accessory minerals in intermediate and basic rocks. In light of this information, we investigated the possible anti-inflammatory and anti-apoptotic effects of clinoptilolite in ADR that is inducing the toxicity in primary liver cell culture. Primary liver cell culture from rat was used in the study. We had three experiment groups including the following: (1) cells treated only with 50 μM ADR for 24 h, (2) cells treated with the 50 μM ADR for 24 h and then treated with 10(-4) M zeolite for 1 h, and (3) cells were incubated with 50 μM ADR for 24 h and then incubated with 10(-4) M zeolite for 24 h to test its long-term effects. After that, western blotting was performed in order to evaluate protein expression levels of several inflammation markers including IL-1β, tumor necrosis factor (TNF)-α, and nuclear factor kappa B (NF-κB), and immunohistochemistry was carried out to detect apoptosis in liver cell culture. Also, TdT-dUTP Terminal Nick-End Labeling (TUNEL) method was used for detecting apoptosis. We found elevated levels of inflammatory protein and apoptotic markers in ADR-administered cells (p < 0.05). Inflammatory and apoptotic markers decreased significantly after treated with zeolite (p < 0.05). The present study was pointed out that ADR causes hepatotoxicity via apoptosis and/or inflammation processes resulting from initiator NF-κB and TNF which causes proinflammatory mediators such as IL-1β. Elevation of inflammation might give rise to trigger apoptosis. Clinoptilolite counteracted the apoptosis and inflammation induced by ADR arising from the

  14. Ceramide mediates the apoptotic response of WEHI 231 cells to anti-immunoglobulin, corticosteroids and irradiation.

    PubMed

    Quintans, J; Kilkus, J; McShan, C L; Gottschalk, A R; Dawson, G

    1994-07-29

    We demonstrate for the first time how immature B cells kill themselves. Ceramide is identified as the mediator of apoptosis in the murine B lymphoma line WEHI 231 commonly used as a model to study clonal deletion in B lymphocytes. We show that exogenous ceramide induces apoptosis in WEHI 231 cells. To maintain self tolerance, immature lymphocytes readily undergo apoptotic death in response to the cross-linking of their antigen-specific receptors. We demonstrate that endogenously produced ceramide accumulates in WEHI 231 cells exposed to anti-IgM, an antigen surrogate before the onset of apoptosis. We also show that two other inducers of apoptosis, irradiation and dexamethasone, cause intracellular accumulation of ceramide. PMID:8048941

  15. The Cytosolic Microbial Receptor Nod2 Regulates Small Intestinal Crypt Damage and Epithelial Regeneration following T Cell-Induced Enteropathy.

    PubMed

    Zanello, Galliano; Goethel, Ashleigh; Rouquier, Sandrine; Prescott, David; Robertson, Susan J; Maisonneuve, Charles; Streutker, Catherine; Philpott, Dana J; Croitoru, Kenneth

    2016-07-01

    Loss of function in the NOD2 gene is associated with a higher risk of developing Crohn's disease (CD). CD is characterized by activation of T cells and activated T cells are involved in mucosal inflammation and mucosal damage. We found that acute T cell activation with anti-CD3 mAb induced stronger small intestinal mucosal damage in NOD2(-/-) mice compared with wild-type mice. This enhanced mucosal damage was characterized by loss of crypt architecture, increased epithelial cell apoptosis, delayed epithelial regeneration and an accumulation of inflammatory cytokines and Th17 cells in the small intestine. Partial microbiota depletion with antibiotics did not decrease mucosal damage 1 d after anti-CD3 mAb injection, but it significantly reduced crypt damage and inflammatory cytokine secretion in NOD2(-/-) mice 3 d after anti-CD3 mAb injection, indicating that microbial sensing by Nod2 was important to control mucosal damage and epithelial regeneration after anti-CD3 mAb injection. To determine which cells play a key role in microbial sensing and regulation of mucosal damage, we engineered mice carrying a cell-specific deletion of Nod2 in villin and Lyz2-expressing cells. T cell activation did not worsen crypt damage in mice carrying either cell-specific deletion of Nod2 compared with wild-type mice. However, increased numbers of apoptotic epithelial cells and higher expression of TNF-α and IL-22 were observed in mice carrying a deletion of Nod2 in Lyz2-expressing cells. Taken together, our results demonstrate that microbial sensing by Nod2 is an important mechanism to regulate small intestinal mucosal damage following acute T cell activation. PMID:27206769

  16. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand

    PubMed Central

    Venkatarame Gowda Saralamma, Venu; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-01-01

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies’ results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer. PMID:26393583

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

    PubMed Central

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

    2016-01-01

    Epithelial–mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway. PMID:26739898

  18. Hypothesis for thermal activation of the caspase cascade in apoptotic cell death at elevated temperatures

    NASA Astrophysics Data System (ADS)

    Pearce, John A.

    2013-02-01

    Apoptosis is an especially important process affecting disease states from HIV-AIDS to auto-immune disease to cancer. A cascade of initiator and executioner capsase functional proteins is the hallmark of apoptosis. When activated the various caspases activate other caspases or cleave structural proteins of the cytoskeleton, resulting in "blebbing" of the plasma membrane forming apoptotic bodies that completely enclose the disassembled cellular components. Containment of the cytosolic components within the apoptotic bodies differentiates apoptosis from necroptosis and necrosis, both of which release fragmented cytosol and other cellular constituents into the intracellular space. Biochemical models of caspase activation reveal the extensive feedback loops characteristic of apoptosis. They clearly explain the failure of Arrhenius models to give accurate predictions of cell survival curves in hyperthermic heating protocols. Nevertheless, each of the individual reaction velocities can reasonably be assumed to follow Arrhenius kinetics. If so, the thermal sensitivity of the reaction velocity to temperature elevation is: ∂k/∂T = Ea [k/RT2]. Particular reaction steps described by higher activation energies, Ea, are likely more thermally-sensitive than lower energy reactions and may initiate apoptosis in the absence of other stress signals. Additionally, while the classical irreversible Arrhenius formulation fails to accurately represent many cell survival and/or dye uptake curves - those that display an early stage shoulder region - an expanded reversible model of the law of mass action equation seems to prove effective and is directly based on a firm theoretical thermodynamic foundation.

  19. 3-Deazaneplanocin A and neplanocin A analogues and their effects on apoptotic cell death.

    PubMed

    Tam, Eric K W; Nguyen, Tuan Minh; Lim, Cheryl Z H; Lee, Puay Leng; Li, Zhimei; Jiang, Xia; Santhanakrishnan, Sridhar; Tan, Tiong Wei; Goh, Yi Ling; Wong, Sze Yue; Yang, Haiyan; Ong, Esther H Q; Hill, Jeffrey; Yu, Qiang; Chai, Christina L L

    2015-01-01

    3-Deazaneplanocin A (DzNep) is a potential epigenetic drug for the treatment of various cancers. DzNep has been reported to deplete histone methylations, including oncogenic EZH2 complex, giving rise to epigenetic modifications that reactivate many silenced tumor suppressors in cancer cells. Despite its promise as an anticancer drug, little is known about the structure-activity relationships of DzNep in the context of epigenetic modifications and apoptosis induction. In this study, a number of analogues of DzNep were examined for DzNep-like ability to induce synergistic apoptosis in cancer cells in combination with trichostatin A, a known histone deacetylase (HDAC) inhibitor. The structure-activity relationship data thus obtained provide valuable information on the structural requirements for biological activity. The studies identified three compounds that show similar activities to DzNep. Two of these compounds show good pharmacokinetics and safety profiles. Attempts to correlate the observed synergistic apoptotic activities with measured S-adenosylhomocysteine hydrolase (SAHH) inhibitory activities suggest that the apoptotic activity of DzNep might not be directly due to its inhibition of SAHH. PMID:25319940

  20. The nuclear receptor Nr4a1 mediates anti-inflammatory effects of apoptotic cells.

    PubMed

    Ipseiz, Natacha; Uderhardt, Stefan; Scholtysek, Carina; Steffen, Martin; Schabbauer, Gernot; Bozec, Aline; Schett, Georg; Krönke, Gerhard

    2014-05-15

    Uptake of apoptotic cells (ACs) by macrophages ensures the nonimmunogenic clearance of dying cells, as well as the maintenance of self-tolerance to AC-derived autoantigens. Upon ingestion, ACs exert an inhibitory influence on the inflammatory signaling within the phagocyte. However, the molecular signals that mediate these immune-modulatory properties of ACs are incompletely understood. In this article, we show that the phagocytosis of apoptotic thymocytes was enhanced in tissue-resident macrophages where this process resulted in the inhibition of NF-κB signaling and repression of inflammatory cytokines, such as IL-12. In parallel, ACs induced a robust expression of a panel of immediate early genes, which included the Nr4a subfamily of nuclear receptors. Notably, deletion of Nr4a1 interfered with the anti-inflammatory effects of ACs in macrophages and restored both NF-κB signaling and IL-12 expression. Accordingly, Nr4a1 mediated the anti-inflammatory properties of ACs in vivo and was required for maintenance of self-tolerance in the murine model of pristane-induced lupus. Thus, our data point toward a key role for Nr4a1 as regulator of the immune response to ACs and of the maintenance of tolerance to "dying self." PMID:24740500

  1. Cytotoxic and apoptotic effects of Ebenus boissieri Barbey on human lung cancer cell line A549

    PubMed Central

    Aydemir, Esra Arslan; Simsek, Ece; Imir, Nilüfer; Göktürk, Ramazan Süleyman; Yesilada, Erdem; Fiskin, Kayahan

    2015-01-01

    Background: Fabaceae family members are known to possess preventive and therapeutic potentials against various types of cancers. Objective: The aim of this study was to investigate the cytotoxic and apoptotic effects of hydroalcoholic extracts from the aerial parts and roots of an endemic Ebenus species; Ebenus boissieri Barbey in human lung cancer cell line. Materials and Methods: After treatment with hydroalcoholic extracts cytotoxic activities of both extracts were measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay, whereas caspase-3 activity, tumor necrosis factor-a lpha (TNF-α) and interferon gamma (IFN-γ) releasewere measured by enzyme linked immunosorbent assay. Results: According to in vitro assay results, the increase in all caspases activity suggested that extracts induce cells to undergo apoptosis. Especially, induction in caspase-3 activity was the most remarkable result of this study. Both aerial part and root extracts induced apoptosis by increasing caspase-3 activity, TNF-α and IFN-γ release. When compared to their relative controls, the concentrations of both TNF-α and IFN-γ in extract-treated groups were significantly and dose dependently exalted. Conclusion: Taken together, our results indicate that the hydroalcoholic extracts of E. boissieri can be considered as a source of new anti-apoptotic and therefore anti-carcinogenic agent. PMID:26109772

  2. Cordycepin enhances cisplatin apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells

    PubMed Central

    Chen, Ying-Hui; Wang, Jo-Yu; Pan, Bo-Syong; Mu, Yi-Fen; Lai, Meng-Shao; So, Edmund Cheung; Wong, Thian-Sze; Huang, Bu-Miin

    2013-01-01

    Purpose The present study aims to investigate whether the combination treatment of cordycepin (an extracted pure compound from Cordyceps sinensis) and cisplatin (a platinum-based chemotherapy drug) has better apoptotic effect in head and neck squamous cell carcinoma (HNSCC). Methods The apoptotic influences of cordycepin and/or cisplatin treatments to human OC3, OEC-M1, and FaDu HNSCC cells were investigated by morphological observations, viability assay, flow cytometry assay, and Western blotting methods. Results Data showed that the cell death phenomenon increased as the dosage of cordycepin or cisplatin increased, and it appeared more in cordycepin plus cisplatin cotreatment among three cell lines. Cell survival rates significantly decreased as the dosage of cordycepin or cisplatin increased, and the better apoptotic effects were observed in cotreatment. Cell cycle analysis further demonstrated that percentages of subG1 cells in cordycepin or cisplatin treatments significantly increased, suggesting that cells underwent apoptosis, and cordycepin plus cisplatin induced many more subG1 cells. Furthermore, cordycepin or cisplatin induced caspase-8, caspase-9, caspase-3, and poly adenosine diphosphate-ribose polymerase protein cleavages, and stimulated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 protein phosphorylations. Moreover, cordycepin plus cisplatin cotreatment significantly activated those proteins with much better effects among three cell lines. Conclusion Cordycepin plus cisplatin have better apoptotic effect by activating caspase activation with possible MAPK pathway involvement in HNSCC cells. PMID:23926438

  3. Cytotoxic and apoptotic activities of Amorphophallus campanulatus tuber extracts against human hepatoma cell line

    PubMed Central

    Ansil, P.N.; Wills, P.J.; Varun, R.; Latha, M.S.

    2014-01-01

    Amorphophallus campanulatus (Roxb.) Blume belonging to the family of Araceae, is a perennial herb commonly known as elephant foot yam. Its tuber has been traditionally used for the treatment of liver diseases, abdominal tumors, piles. The aim of the present study was to evaluate the dose-dependent cytotoxic and apoptosis inducing effects of the sub fractions of Amorphophallus campanulatus tuber methanolic extract (ACME) namely petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF) and methanolic fraction (MeF) on human liver cancer cell line, PLC/PRF/5. Antiproliferative effects of the sub fractions of ACME were studied by MTT assay. Apoptotic activity was assessed by 4′,6-diamidino-2-phenylindole (DAPI), annexin V- fluorescein isothiocyanate (FITC) and 5,5’,6,6’ tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescent staining. The chemotherapeutic drug, 5-flurouracil (5-FU) was used as positive drug control. The sub fractions of ACME were found to produce considerable cytotoxicity in human liver cancer cell line, PLC/PRF/5. In addition, the extracts were found to induce apoptosis and were substantiated by DAPI, annexin V-FITC and JC-1 fluorescent staining. A pronounced results of cytotoxic and apoptotic activities were observed in the cells treated with 5-FU and CHF, whereas, EAF and MeF treated cells exhibited a moderate result and the least effect were observed in PEF treated cells. Furthermore, these findings confirm that the sub fractions of ACME dose-dependently suppress the proliferation of PLC/PRF/5 cells by inducing apoptosis. PMID:25657798

  4. Cytotoxic and apoptotic activities of Amorphophallus campanulatus tuber extracts against human hepatoma cell line.

    PubMed

    Ansil, P N; Wills, P J; Varun, R; Latha, M S

    2014-01-01

    Amorphophallus campanulatus (Roxb.) Blume belonging to the family of Araceae, is a perennial herb commonly known as elephant foot yam. Its tuber has been traditionally used for the treatment of liver diseases, abdominal tumors, piles. The aim of the present study was to evaluate the dose-dependent cytotoxic and apoptosis inducing effects of the sub fractions of Amorphophallus campanulatus tuber methanolic extract (ACME) namely petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF) and methanolic fraction (MeF) on human liver cancer cell line, PLC/PRF/5. Antiproliferative effects of the sub fractions of ACME were studied by MTT assay. Apoptotic activity was assessed by 4',6-diamidino-2-phenylindole (DAPI), annexin V- fluorescein isothiocyanate (FITC) and 5,5',6,6' tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescent staining. The chemotherapeutic drug, 5-flurouracil (5-FU) was used as positive drug control. The sub fractions of ACME were found to produce considerable cytotoxicity in human liver cancer cell line, PLC/PRF/5. In addition, the extracts were found to induce apoptosis and were substantiated by DAPI, annexin V-FITC and JC-1 fluorescent staining. A pronounced results of cytotoxic and apoptotic activities were observed in the cells treated with 5-FU and CHF, whereas, EAF and MeF treated cells exhibited a moderate result and the least effect were observed in PEF treated cells. Furthermore, these findings confirm that the sub fractions of ACME dose-dependently suppress the proliferation of PLC/PRF/5 cells by inducing apoptosis. PMID:25657798

  5. Biomarkers distinguish apoptotic and necrotic cell death during hepatic ischemia/reperfusion injury in mice.

    PubMed

    Yang, Min; Antoine, Daniel J; Weemhoff, James L; Jenkins, Rosalind E; Farhood, Anwar; Park, B Kevin; Jaeschke, Hartmut

    2014-11-01

    Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin-stained and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (≤3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to

  6. BIOMARKERS DISTINGUISH APOPTOTIC AND NECROTIC CELL DEATH DURING HEPATIC ISCHEMIA-REPERFUSION INJURY IN MICE

    PubMed Central

    Yang, Min; Antoine, Daniel J.; Weemhoff, James L.; Jenkins, Rosalind E.; Farhood, Anwar; Park, B. Kevin; Jaeschke, Hartmut

    2014-01-01

    Hepatic ischemia-reperfusion (IRP) injury is a significant clinical problem during tumor resection surgery (Pringle maneuver), and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. In order to address this important issue in a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA-122, full-length cytokeratin-18 (FK18) and high mobility group box-1 (HMGB1) protein, and apoptosis including plasma caspase-3 activity and caspase-cleaved cyto