Exogenous ACE2 Expression Allows Refractory Cell Lines To Support Severe Acute Respiratory Syndrome Coronavirus Replication

PubMed Central

Mossel, Eric C.; Huang, Cheng; Narayanan, Krishna; Makino, Shinji; Tesh, Robert B.; Peters, C. J.

2005-01-01

Of 30 cell lines and primary cells examined, productive severe acute respiratory syndrome coronavirus (Urbani strain) (SARS-CoV) infection after low-multiplicity inoculation was detected in only six: three African green monkey kidney epithelial cell lines (Vero, Vero E6, and MA104), a human colon epithelial line (CaCo-2), a porcine kidney epithelial line [PK(15)], and mink lung epithelial cells (Mv 1 Lu). SARS-CoV produced a lytic infection in Vero, Vero E6, and MA104 cells, but there was no visible cytopathic effect in Caco-2, Mv 1 Lu, or PK(15) cells. Multistep growth kinetics were identical in Vero E6 and MA104 cells, with maximum titer reached 24 h postinoculation (hpi). Virus titer was maximal 96 hpi in CaCo-2 cells, and virus was continually produced from infected CaCo-2 cells for at least 6 weeks after infection. CaCo-2 was the only human cell type of 13 tested that supported efficient SARS-CoV replication. Expression of the SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), resulted in SARS-CoV replication in all refractory cell lines examined. Titers achieved were variable and dependent upon the method of ACE2 expression. PMID:15731278

Productive infection of Epstein-Barr virus (EBV) in EBV-genome-positive epithelial cell lines (GT38 and GT39) derived from gastric tissues.

PubMed

Takasaka, N; Tajima, M; Okinaga, K; Satoh, Y; Hoshikawa, Y; Katsumoto, T; Kurata, T; Sairenji, T

1998-08-01

We characterized the expression of Epstein-Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF 1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA 1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or n-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cells lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with a Xhol probe in both cell lines. These epithelial cells lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

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

PubMed

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

2009-04-01

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

Extremely stringent activation of p16INK4a prevents immortalization of uterine cervical epithelial cells without human papillomavirus oncogene expression

PubMed Central

Hang, Su; Tiwari, Agnes F.Y.; Ngan, Hextan Y.S.; Yip, Yim-Ling; Cheung, Annie L.M.; Tsao, Sai Wah; Deng, Wen

2016-01-01

Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16INK4a, which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer. PMID:27344169

Characterization of newly established bovine intestinal epithelial cell line.

PubMed

Miyazawa, Kohtaro; Hondo, Tetsuya; Kanaya, Takashi; Tanaka, Sachi; Takakura, Ikuro; Itani, Wataru; Rose, Michael T; Kitazawa, Haruki; Yamaguchi, Takahiro; Aso, Hisashi

2010-01-01

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer's patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.

Lactobacillus acidophilus Induces Cytokine and Chemokine Production via NF-κB and p38 Mitogen-Activated Protein Kinase Signaling Pathways in Intestinal Epithelial Cells

PubMed Central

Lü, Xuena; Man, Chaoxin; Han, Linlin; Shan, Yi; Qu, Xingguang; Liu, Ying; Yang, Shiqin; Xue, Yuqing; Zhang, Yinghua

2012-01-01

Intestinal epithelial cells can respond to certain bacteria by producing an array of cytokines and chemokines which are associated with host immune responses. Lactobacillus acidophilus NCFM is a characterized probiotic, originally isolated from human feces. This study aimed to test the ability of L. acidophilus NCFM to stimulate cytokine and chemokine production in intestinal epithelial cells and to elucidate the mechanisms involved in their upregulation. In experiments using intestinal epithelial cell lines and mouse models, we observed that L. acidophilus NCFM could rapidly but transiently upregulate a number of effector genes encoding cytokines and chemokines such as interleukin 1α (IL-1α), IL-1β, CCL2, and CCL20 and that cytokines showed lower expression levels with L. acidophilus NCFM treatment than chemokines. Moreover, L. acidophilus NCFM could activate a pathogen-associated molecular pattern receptor, Toll-like receptor 2 (TLR2), in intestinal epithelial cell lines. The phosphorylation of NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) in intestinal epithelial cell lines was also enhanced by L. acidophilus NCFM. Furthermore, inhibitors of NF-κB (pyrrolidine dithiocarbamate [PDTC]) and p38 MAPK (SB203580) significantly reduced cytokine and chemokine production in the intestinal epithelial cell lines stimulated by L. acidophilus NCFM, suggesting that both NF-κB and p38 MAPK signaling pathways were important for the production of cytokines and chemokines induced by L. acidophilus NCFM. PMID:22357649

Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells.

PubMed

Thomas, Richard J; Brooks, Tim J

2004-02-01

Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.

Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing.

PubMed

Lin, Louis M; Huang, George T-J; Rosenberg, Paul A

2007-08-01

There is continuing controversy regarding the potential for inflammatory apical cysts to heal after nonsurgical endodontic therapy. Molecular cell biology may provide answers to a series of related questions. How are the epithelial cell rests of Malassez stimulated to proliferate? How are the apical cysts formed? How does the lining epithelium of apical cysts regress after endodontic therapy? Epithelial cell rests are induced to divide and proliferate by inflammatory mediators, proinflammatory cytokines, and growth factors released from host cells during periradicular inflammation. Quiescent epithelial cell rests can behave like restricted-potential stem cells if stimulated to proliferate. Formation of apical cysts is most likely caused by the merging of proliferating epithelial strands from all directions to form a three-dimensional ball mass. After endodontic therapy, epithelial cells in epithelial strands of periapical granulomas and the lining epithelium of apical cysts may stop proliferating because of a reduction in inflammatory mediators, proinflammatory cytokines, and growth factors. Epithelial cells will also regress because of activation of apoptosis or programmed cell death through deprivation of survival factors or by receiving death signals during periapical wound healing.

Continuous human cell lines and method of making same

DOEpatents

Stampfer, Martha R.

1989-01-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT).

PubMed

Chen, Li-Mei; Verity, Nicole J; Chai, Karl X

2009-10-22

The glycosylphosphatidylinositol (GPI)-anchored epithelial extracellular membrane serine protease prostasin (PRSS8) is expressed abundantly in normal epithelia and essential for terminal epithelial differentiation, but down-regulated in human prostate, breast, and gastric cancers and invasive cancer cell lines. Prostasin is involved in the extracellular proteolytic modulation of the epidermal growth factor receptor (EGFR) and is an invasion suppressor. The aim of this study was to evaluate prostasin expression states in the transitional cell carcinomas (TCC) of the human bladder and in human TCC cell lines. Normal human bladder tissues and TCC on a bladder cancer tissue microarray (TMA) were evaluated for prostasin expression by means of immunohistochemistry. A panel of 16 urothelial and TCC cell lines were evaluated for prostasin and E-cadherin expression by western blot and quantitative PCR, and for prostasin gene promoter region CpG methylation by methylation-specific PCR (MSP). Prostasin is expressed in the normal human urothelium and in a normal human urothelial cell line, but is significantly down-regulated in high-grade TCC and lost in 9 (of 15) TCC cell lines. Loss of prostasin expression in the TCC cell lines correlated with loss of or reduced E-cadherin expression, loss of epithelial morphology, and promoter DNA hypermethylation. Prostasin expression could be reactivated by demethylation or inhibition of histone deacetylase. Re-expression of prostasin or a serine protease-inactive variant resulted in transcriptional up-regulation of E-cadherin. Loss of prostasin expression in bladder transitional cell carcinomas is associated with epithelial-mesenchymal transition (EMT), and may have functional implications in tumor invasion and resistance to chemotherapy.

The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

PubMed

Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

2017-04-06

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

PubMed

Fu, Mei; Chen, Yabing; Xiong, Xianrong; Lan, Daoliang; Li, Jian

2014-01-01

This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

NASA Astrophysics Data System (ADS)

Herceg, Zdenko

Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the basic epithelial morphology of the parent HToriS cells. Investigation of radiosensitivity showed that none of the 6 tumour cell lines examined had a higher radiosensitivity compared to the parent HToriS cells. This excludes the possibility that the observed transformation was the result of the selection of a pre-existing transformed subpopulation of the parent cells but that radiation-induced transformants were being induced de novo. The tumour cell lines were screened for mutations in H- and K-ras oncogenes using restriction enzyme analysis of PCR amplified DNA. No mutations were detected in 26 tumour cell lines suggesting that mutations in these two genes do not appear to be involved in radiation- induced neoplastic transformation in human thyroid epithelial cells. Screening for mutations in p53 protein using immunoprecipitation method detected no mutations in 6 tumour cell lines. This human thyroid epithelial cell line may thus be useful for the in vitro study of cellular and molecular mechanisms that are involved in human epithelial cell carcinogenesis.

Continuous human cell lines and method of making same

DOEpatents

Stampfer, M.R.

1985-07-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line.

PubMed

Tong, Hui-Li; Li, Qing-Zhang; Gao, Xue-Jun; Yin, De-Yun

2012-03-01

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.

Vitamin D3 analog maxacalcitol (OCT) induces hCAP-18/LL-37 production in human oral epithelial cells.

PubMed

Tada, Hiroyuki; Shimizu, Takamitsu; Nagaoka, Isao; Takada, Haruhiko

2016-01-01

Maxacalcitol (22-oxacalcitriol: OCT) is a synthetic vitamin D3 analog with a limited calcemic effect. In this study, we investigated whether OCT increases the production of LL-37/CAP-18, a human cathelicidin antimicrobial peptide, in human gingival/oral epithelial cells. A human gingival epithelial cell line (Ca9-22) and human oral epithelial cell lines (HSC-2, HSC-3, and HSC-4) exhibited the enhanced expression of LL-37 mRNA upon stimulation with OCT as well as active metabolites of vitamins D3 and D2. Among the human epithelial cell lines, Ca9-22 exhibited the strongest response to these vitamin D-related compounds. OCT induced the higher production of CAP-18 (ng/mL order) until 6 days time-dependently in Ca9-22 cells in culture. The periodontal pathogen Porphyromonas gingivalis was killed by treatment with the LL-37 peptide. These findings suggest that OCT induces the production of hCAP-18/LL-37 in a manner similar to that induced by the active metabolite of vitamin D3.

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

PubMed

Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

2006-02-17

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells.

PubMed

Reyes-Reyes, Elsa M; Aispuro, Ivan; Tavera-Garcia, Marco A; Field, Matthew; Moore, Sara; Ramos, Irma; Ramos, Kenneth S

2017-11-28

Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo . These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

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

PubMed

Sweat JMDunigan, D D; Wright, S D

2001-06-01

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

Identification of Novel Prognostic Genetic Markers in Prostate Cancer

DTIC Science & Technology

2000-02-01

alterations in two normal- and three malignant-derived prostate epithelial cell lines immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV...malignant-derived prostate epithelial cell lines immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV) 16. These studies...transforming genes of human papilloma virus (HPV) 16 (13). The cell lines demonstrated several numerical and structural chromosomal alterations

Invasion of Human Oral Epithelial Cells by Prevotella intermedia

PubMed Central

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

1998-01-01

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

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

PubMed

Lee, Jonathan K; Garbe, James C; Vrba, Lukas; Miyano, Masaru; Futscher, Bernard W; Stampfer, Martha R; LaBarge, Mark A

2015-01-01

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

Study designs to investigate Nox1 acceleration of neoplastic progression in immortalized human epithelial cells by selection of differentiation resistant cells.

PubMed

Sattayakhom, Apsorn; Chunglok, Warangkana; Ittarat, Wanida; Chamulitrat, Walee

2014-01-01

To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines - FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4[S

PubMed Central

Lukic, Ana; Ji, Jie; Idborg, Helena; Samuelsson, Bengt; Palmberg, Lena

2016-01-01

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4. In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4. Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung. PMID:27436590

[Expression of Chemokine receptor CXCR6 and its significance in breast cancer cell lines].

PubMed

Cheng, Hao; Chen, Nian-yong

2014-05-01

To detect the expression of Chemokine receptor CXCR6 in invasive breast cancer cell lines and normal mammary epithelial cell line, and assess the relationship between CXCR6 expression and malignant behavior of breast cancer cells. Expression level of CXCR6 in different invasive breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-231) and normal mammary epithelial cell line (MCF-10A)was detected by real time reverse transcription-polymerase chain reaction (real time-PCR) and Western blot. Lentivirus was employed to interfere CXCR6 expression in MDA-MB-231. MTT assay and transwell chamber were used to study proliferative and invasive ability of those cells respectively. Vascular enothelial growth factor (VEGF) expression was detected to study the role of CXCR6 in angiogenesis. At both mRNA level and protein level, normal mammary epithelial cell line MCF-10A showed the weakest CXCR6 expression. The breast cancer cell lines expressed CXCR6 in different levels, the expression level of CXCR6 in highly invasive cell line MDA-MB-231 was significantly higher than that in two low-invasive cell lines SK-BR-3 and MCF-7 (P < 0.05). Silencing CXCR6 gene by Lentivirus-mediated RNA interference in MDA-MB-231 inhibited its proliferation ability, invasion ability and angiogenesis ability in vitro (P < 0.05). Different invasive breast cancer cell lines express CXCR6 at different levels, positively correlated with its invasive ability.

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

PubMed Central

Liévin-Le Moal, Vanessa

2013-01-01

SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

PubMed Central

Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

2016-01-01

Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

Apical Cyst Theory: a Missing Link.

PubMed

Huang, George T-J

2010-10-05

The mechanism of the formation of apical cyst has been elusive. Several theories have long been proposed and discussed speculating how an apical cyst is developed and formed in the jaw bone resulting from endododontic infection. Two popular theories are the nutritional deficiency theory and the abscess theory. The nutritional deficiency theory assumes that the over proliferated epithelial cells will form a ball mass such that the cells in the center of the mass will be deprived of nutrition. The abscess theory postulates that when an abscess cavity is formed in connective tissue, epithelial cells proliferate and line the preexisting cavity because of their inherent tendency to cover exposed connective tissue surfaces. Based on the nature of epithelial cells and the epithelium, nutritional theory is a fairy tale, while abscess theory at best just indicates that abscess may be one of the factors that allows the stratified epithelium to form but not to explain a mechanism that makes the cyst to form. Apical cyst formation is the result of proliferation of resting epithelial cells, due to inflammation, to a sufficient number such that they are able to form a polarized and stratified epithelial lining against dead tissues or foreign materials. These stratified epithelial lining expands along the dead tissue or foreign materials and eventually wrap around them as a spherical sac, i.e. a cyst. The space in the sac is considered the external environment separating the internal (tissue) environment - the natural function of epithelium. This theory may be tested by introducing a biodegradable device able to slowly release epithelial cell mitogens in an in vivo environment implanted with epithelial cells next to a foreign object. This will allow the cells to continuously proliferate which may form a cystic sac wrapping around the foreign object.

Apical Cyst Theory: a Missing Link

PubMed Central

Huang, George T.-J.

2012-01-01

Introduction The mechanism of the formation of apical cyst has been elusive. Several theories have long been proposed and discussed speculating how an apical cyst is developed and formed in the jaw bone resulting from endododontic infection. Two popular theories are the nutritional deficiency theory and the abscess theory. The nutritional deficiency theory assumes that the over proliferated epithelial cells will form a ball mass such that the cells in the center of the mass will be deprived of nutrition. The abscess theory postulates that when an abscess cavity is formed in connective tissue, epithelial cells proliferate and line the preexisting cavity because of their inherent tendency to cover exposed connective tissue surfaces. Based on the nature of epithelial cells and the epithelium, nutritional theory is a fairy tale, while abscess theory at best just indicates that abscess may be one of the factors that allows the stratified epithelium to form but not to explain a mechanism that makes the cyst to form. The hypothesis Apical cyst formation is the result of proliferation of resting epithelial cells, due to inflammation, to a sufficient number such that they are able to form a polarized and stratified epithelial lining against dead tissues or foreign materials. These stratified epithelial lining expands along the dead tissue or foreign materials and eventually wrap around them as a spherical sac, i.e. a cyst. The space in the sac is considered the external environment separating the internal (tissue) environment – the natural function of epithelium. Evaluation of the hypothesis This theory may be tested by introducing a biodegradable device able to slowly release epithelial cell mitogens in an in vivo environment implanted with epithelial cells next to a foreign object. This will allow the cells to continuously proliferate which may form a cystic sac wrapping around the foreign object. PMID:25346864

Cytokeratin 5 positive cells represent a therapy resistant subpopulation in epithelial ovarian cancer

PubMed Central

Corr, Bradley R.; Finlay-Schultz, Jessica; Rosen, Rachel B.; Qamar, Lubna; Post, Miriam D.; Behbakht, Kian; Spillman, Monique A.; Sartorius, Carol A.

2015-01-01

Objective Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)+ breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5+ cell populations to cisplatin therapy. Materials and Methods CK5 expression was evaluated in two ovarian tissue microarrays, representing 137 neoplasms, and six ovarian cancer cell lines. Cell lines were treated with IC50 cisplatin and the prevalence of CK5+ cells pre- and post-treatment determined. Proliferation of CK5+ vs. CK5− cell populations was determined using bromodeoxyuridine (BrdU) incorporation. Chemotherapy induced apoptosis in CK5+ vs. CK5− cells was measured using immunohistochemical staining for cleaved caspase-3. Results CK5 was expressed in 39.3% (42/107) of epithelial ovarian cancers with a range of 1-80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5+ specimens. CK5 expression correlated with ER positivity (38/42 CK5+ tumors were also ER+). CK5 was expressed in 5/6 overall and 4/4 ER+ epithelial ovarian cancer cell lines ranging from 2.4-52.7% positive cells. CK5+ compared to CK5− cells were slower proliferating. The prevalence of CK5+ cells increased following 48 hour cisplatin treatment in 4/5 cell lines tested. CK5+ compared to CK5− ovarian cancer cells were more resistant to cisplatin induced apoptosis. Conclusions CK5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating, chemoresistant subpopulation that may warrant co-targeting in combination therapy. PMID:26495758

RBFOX2 Is an Important Regulator of Mesenchymal Tissue-Specific Splicing in both Normal and Cancer Tissues

PubMed Central

Venables, Julian P.; Brosseau, Jean-Philippe; Gadea, Gilles; Klinck, Roscoe; Prinos, Panagiotis; Beaulieu, Jean-François; Lapointe, Elvy; Durand, Mathieu; Thibault, Philippe; Tremblay, Karine; Rousset, François; Tazi, Jamal; Abou Elela, Sherif

2013-01-01

Alternative splicing provides a critical and flexible layer of regulation intervening in many biological processes to regulate the diversity of proteins and impact cell phenotype. To identify alternative splicing differences that distinguish epithelial from mesenchymal tissues, we have investigated hundreds of cassette exons using a high-throughput reverse transcription-PCR (RT-PCR) platform. Extensive changes in splicing were noted between epithelial and mesenchymal tissues in both human colon and ovarian tissues, with many changes from mostly one splice variant to predominantly the other. Remarkably, many of the splicing differences that distinguish normal mesenchymal from normal epithelial tissues matched those that differentiate normal ovarian tissues from ovarian cancer. Furthermore, because splicing profiling could classify cancer cell lines according to their epithelial/mesenchymal characteristics, we used these cancer cell lines to identify regulators for these specific splicing signatures. By knocking down 78 potential splicing factors in five cell lines, we provide an extensive view of the complex regulatory landscape associated with the epithelial and mesenchymal states, thus revealing that RBFOX2 is an important driver of mesenchymal tissue-specific splicing. PMID:23149937

Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

NASA Technical Reports Server (NTRS)

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

1997-01-01

Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

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

Cyclic adenosine monophosphate modulates cell morphology and behavior of a cultured renal epithelial.

PubMed

Amsler, K

1990-07-01

The role of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) in modulating functions of differentiated renal cells is well established. Its importance in controlling their growth and differentiation is less clear. We have used somatic cell genetic techniques to probe the role of PKA in controlling morphology and behavior of a renal epithelial cell line, LLC-PK1, which acquires many properties characteristic of the renal proximal tubular cell. Mutants of this line altered in PKA activity have been isolated and their behavior compared to that of the parent line. The results indicate that PKA is involved, either directly or indirectly, in maintenance of cell morphology, cell-cell and cell-substratum interactions, density-dependent growth regulation, and expression of one function characteristic of the renal proximal tubular cell, Na-hexose symport. The relevance of these results to the role of PKA in controlling growth and differentiation of renal epithelial cells in vivo is discussed.

Reversible transition towards a fibroblastic phenotype in a rat carcinoma cell line.

PubMed

Boyer, B; Tucker, G C; Vallés, A M; Gavrilovic, J; Thiery, J P

1989-01-01

Two distinct mechanisms by which bladder carcinoma cells of the NBT-II cell line dissociate and migrate away from an in vitro reconstituted epithelial sheet were examined as regards intercellular adhesion and cell locomotion. Scattering of NBT-II bladder carcinoma cell line was promoted by 2 distinct culture protocols: (i) deposition of some components of the extracellular matrix onto the culture substratum (glass or plastic) induced cell dispersion of the epithelial sheet of carcinoma cells, and (ii) addition of Ultroser G, a serum substitute, to the culture medium induced scattering and acquisition of motility of NBT-II cells. Under both culture conditions, NBT-II cells dissociated, lost their epithelial morphology, acquired fibroblastic shape and migrated actively. We show that, among different extracellular matrix proteins, only collagens were able to promote the transition towards fibroblastic phenotype (referred as epithelium-to-mesenchyme transition or EMT). Furthermore, the native 3-dimensional helical structure of collagens was required for their function. During induction of EMT of NBT-II cells with Ultroser G, the junctions between epithelial cells were split, polarized epithelial cell organization was lost, and the resulting individual cells became motile and assumed a spindle-like fibroblastoid appearance. Using immunofluorescence microscopy techniques, we demonstrate that this change is accompanied by redistribution of desmosomal plaque proteins (desmoplakins, desmoglein, plakoglobin) and by reorganization of the cytokeratin and the actin-fodrin filament systems. Intermediate-sized filaments of the vimentin type were formed de novo in the fibroblastoid cell form. The observed transition towards fibroblastic phenotype (epithelium-to-mesenchyme transition or EMT) was fully reversed by removing the inducing factors from the culture medium, as shown by the disappearance of vimentin filaments and the reappearance of desmosomes in the newly formed epithelial cells.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines.

PubMed

Syed, H Claudia; Dubreuil, J Daniel

2012-09-01

A previous study conducted in our laboratory demonstrated that cells having internalized Escherichia coli STb toxin display apoptotic-like morphology. We therefore investigated if STb could induce programmed cell death in both a human and an animal intestinal epithelial cell lines. HRT-18 (Human Colon Tumor) and IEC-18 (Rat Ileum Epithelial Cells) cell lines were used. As STb is frequently tested in a rat model, the IEC-18 cell line was most relevant to our work. The cell lines were treated with various amounts of purified STb (nanomole range) for a period of 24 h after which cells were harvested and examined for apoptotic characteristics. Caspase-9, the initiator of mitochondrion-mediated apoptosis, and caspase-3, an effector of caspase-9, were both activated following STb intoxication of HRT-18 and IEC-18 cells whereas caspase-8, the initiator caspase of the extrinsic pathway, was not activated. For both cell lines, agarose gel electrophoresis of the cell DNA content reveals laddering of DNA, resulting from DNA fragmentation, a characteristic of apoptosis. Hoechst 33342-stained DNA of STb-treated cell lines, observed using fluorescence microscopy, revealed condensation and fragmentation of the nuclei. Apoptotic indexes calculated from fragmented nuclei of Hoechst 33342-stained DNA for HRT-18 and IEC-18 cells showed an STb dose-dependent response. Overall, these data indicate that STb toxin induces a mitochondrion-mediated caspase-dependent apoptotic pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

Stably Fluorescent Cell Line of Human Ovarian Epithelial Cancer Cells SK-OV-3ip-red.

PubMed

Konovalova, E V; Shulga, A A; Chumakov, S P; Khodarovich, Yu M; Woo, Eui-Jeon; Deev, S M

2017-11-01

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

A critical synopsis: Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

NASA Technical Reports Server (NTRS)

Chuman, L. M.; FINE; COHEN; Saier, M. H.

1985-01-01

The kidney forms urine and reabsorbs electrolytes and water. Kidney cell lines and hormone supplemented serum free medium were used for growth. The hormones were insulin, transferrin, vasopressin, cholesterol, prostaglandins, hydrocortisone, and triidothyronine. Epithelial cell lines are polar and form hemicysts. The Madin-Darby canine kidney(MDCK) cell line used is distal tubulelike. LLC-PK sub 1 cells are derived from pig kidneys and have the properties of different kidney segments. The LLC-PK sub 1 cells with proximal tubule properties were maintained in hormone-supplemented serum free medium. Seven factors (the aforementioned homrones and selenium) were needed for growth. Hormone-defined medium supported LLC-PK sub 1 cell growth, allowed transport (as seen by hemicyst formation), and influenced cell morphology. Vasopressin (used for growth and morphology) could be partially replaced by isobutylmethylxanthine or dibutyryl cAMP. The defined medium was used to isolate rabbit proximal tubule kidney epithelial cells free of fibroblasts.

Tumour necrosis factor (TNF)-mediated NF-κB activation facilitates cellular invasion of non-professional phagocytic epithelial cell lines by Trypanosoma cruzi.

PubMed

Pinto, Andrea M T; Sales, Paula C M; Camargos, Elizabeth R S; Silva, Aristóbolo M

2011-10-01

At the site of infection, pro-inflammatory cytokines locally produced by macrophages infected with Trypanosoma cruzi can activate surrounding non-professional phagocytes such as fibroblasts, epithelial and endothelial cells, which can be further invaded by the parasite. The effect of secreted soluble factors on the invasion of these cells remains, however, to be established. We show here that two epithelial cell lines become significantly susceptible to the infection by the Y strain of T. cruzi after tumour necrosis factor (TNF) treatment. The increase in the invasion was correlated with the increasing concentration of recombinant TNF added to cultures of HEK293T or LLC-MK2 cells. Supernatants taken from PMA-differentiated human monocytes infected with T. cruzi also increased the permissiveness of epithelial cells to subsequent infection with the parasite, which was inhibited by a TNF monoclonal antibody. Furthermore, the permissiveness induced by TNF was inhibited by TPCK, and led to significant decrease in the number of intracellular parasites, providing evidence that activation of NF-κB induced by TNF favours the invasion of the epithelial cell lines by T. cruzi through yet an unidentified mechanism. Our data indicate that soluble factors released from macrophages early in the infection favours T. cruzi invasion of non-professional phagocytic cells. © 2011 Blackwell Publishing Ltd.

Efficacy of neratinib in the treatment of HER2/neu-amplified epithelial ovarian carcinoma in vitro and in vivo.

PubMed

Menderes, Gulden; Bonazzoli, Elena; Bellone, Stefania; Black, Jonathan D; Lopez, Salvatore; Pettinella, Francesca; Masserdotti, Alice; Zammataro, Luca; Litkouhi, Babak; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D

2017-05-01

Epithelial ovarian carcinoma is the most lethal of gynecologic malignancies. There is a need to optimize the currently available treatment strategies and to urgently develop novel therapeutic agents against chemotherapy-resistant disease. The objective of our study was to evaluate neratinib's preclinical efficacy in treating HER2-amplified ovarian cancer. Neratinib's efficacy in treating HER2-amplified ovarian cancer was studied in vitro utilizing six primary tumor cell lines with differential HER2/neu expression. Flow cytometry was utilized to assess IC 50 , cell signaling changes, and cell cycle distribution. Neratinib's in vivo efficacy was evaluated in HER2-amplified epithelial ovarian carcinoma xenografts. Three of six (50%) ovarian cancer cell lines were HER2/neu-amplified. Neratinib showed significantly higher efficacy in treating HER2/neu-amplified cell lines when compared to the non-HER2/neu-amplified tumor cell lines (mean ± SEM IC 50 :0.010 μM ± 0.0003 vs. 0.076 μM ± 0.005 p < 0.0001). Neratinib treatment significantly decreased the phosphorylation of the transcription factor S6, leading to arrest of the cell cycle in G0/G1 phase. Neratinib prolonged survival in mice harboring HER2-amplified epithelial ovarian carcinoma xenografts (p = 0.003). Neratinib inhibits proliferation, signaling, cell cycle progression and tumor growth of HER2-amplified epithelial ovarian carcinoma in vitro. Neratinib inhibits xenograft growth and improves overall survival in HER2/neu-amplified ovarian cancer in vivo. Clinical trials are warranted.

Efficacy of neratinib in the treatment of HER2/neu-amplified epithelial ovarian carcinoma in vitro and in vivo

PubMed Central

Menderes, Gulden; Bonazzoli, Elena; Bellone, Stefania; Black, Jonathan D.; Lopez, Salvatore; Pettinella, Francesca; Masserdotti, Alice; Zammataro, Luca; Litkouhi, Babak; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E.

2018-01-01

Epithelial ovarian carcinoma is the most lethal of gynecologic malignancies. There is a need to optimize the currently available treatment strategies and to urgently develop novel therapeutic agents against chemotherapy-resistant disease. The objective of our study was to evaluate neratinib’s preclinical efficacy in treating HER2-amplified ovarian cancer. Neratinib’s efficacy in treating HER2-amplified ovarian cancer was studied in vitro utilizing six primary tumor cell lines with differential HER2/neu expression. Flow cytometry was utilized to assess IC50, cell signaling changes, and cell cycle distribution. Neratinib’s in vivo efficacy was evaluated in HER2-amplified epithelial ovarian carcinoma xenografts. Three of six (50%) ovarian cancer cell lines were HER2/neu-amplified. Neratinib showed significantly higher efficacy in treating HER2/neu-amplified cell lines when compared to the non-HER2/neu-amplified tumor cell lines (mean ± SEM IC50:0.010 μM ± 0.0003 vs. 0.076 μM ± 0.005 p < 0.0001). Neratinib treatment significantly decreased the phosphorylation of the transcription factor S6, leading to arrest of the cell cycle in G0/G1 phase. Neratinib prolonged survival in mice harboring HER2-amplified epithelial ovarian carcinoma xenografts (p = 0.003). Neratinib inhibits proliferation, signaling, cell cycle progression and tumor growth of HER2-amplified epithelial ovarian carcinoma in vitro. Neratinib inhibits xenograft growth and improves overall survival in HER2/neu-amplified ovarian cancer in vivo. Clinical trials are warranted. PMID:28397106

Isolation of Mouse Primary Gastric Epithelial Cells to Investigate the Mechanisms of Helicobacter pylori Associated Disease.

PubMed

Tran, Le Son; Ferrero, Richard L

2018-01-01

The gastrointestinal epithelium provides the first line of defense against invading pathogens, among which Helicobacter pylori is linked to numerous gastric pathologies, including chronic gastritis and cancer. Primary gastric epithelial cells represent a useful model for the investigation of the underlying molecular and cellular mechanisms involved in these H. pylori associated diseases. In this chapter, we describe a method for the isolation of primary gastric epithelial cells from mice and detection of epithelial cell adhesion molecule (EpCAM) expression in the isolated cells.

Development and characterization of a cell line WAF from freshwater shark Wallago attu.

PubMed

Dubey, Akhilesh; Goswami, Mukunda; Yadav, Kamalendra; Sharma, Bhagwati S

2014-02-01

A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants.

DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

PubMed

Houshdaran, Sahar; Hawley, Sarah; Palmer, Chana; Campan, Mihaela; Olsen, Mari N; Ventura, Aviva P; Knudsen, Beatrice S; Drescher, Charles W; Urban, Nicole D; Brown, Patrick O; Laird, Peter W

2010-02-22

Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential tumor progenitor cells, which may help illuminate the etiology and natural history of these cancers.

Cellular Migration and Invasion Uncoupled: Increased Migration Is Not an Inexorable Consequence of Epithelial-to-Mesenchymal Transition

PubMed Central

Schaeffer, Daneen; Somarelli, Jason A.; Hanna, Gabi; Palmer, Gregory M.

2014-01-01

Metastatic dissemination requires carcinoma cells to detach from the primary tumor and invade through the basement membrane. To acquire these characteristics, epithelial tumor cells undergo epithelial-to-mesenchymal transitions (EMT), whereby cells lose polarity and E-cadherin-mediated cell-cell adhesion. Post-EMT cells have also been shown, or assumed, to be more migratory; however, there have been contradictory reports on an immortalized human mammary epithelial cell line (HMLE) that underwent EMT. In the context of carcinoma-associated EMT, it is not yet clear whether the change in migration and invasion must be positively correlated during EMT or whether enhanced migration is a necessary consequence of having undergone EMT. Here, we report that pre-EMT rat prostate cancer (PC) and HMLE cells are more migratory than their post-EMT counterparts. To determine a mechanism for increased epithelial cell migration, gene expression analysis was performed and revealed an increase in epidermal growth factor receptor (EGFR) expression in pre-EMT cells. Indeed, inhibition of EGFR in PC epithelial cells slowed migration. Importantly, while post-EMT PC and HMLE cell lines are less migratory, both remain invasive in vitro and, for PC cells, in vivo. Our study demonstrates that enhanced migration is not a phenotypic requirement of EMT, and migration and invasion can be uncoupled during carcinoma-associated EMT. PMID:25002532

The Role of the Rab Coupling Protein in ErbB2-Driven Mammary Tumorigenesis and Metastasis

DTIC Science & Technology

2014-10-01

Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation, Senescence. 16. SECURITY CLASSIFICATION OF: 17...Tyrosine Kinase, Her/ErbB2 signaling, Rab Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation...lines included RCP condition to internalization and detection of E-cadherin, a well-known adherent junction and epithelial mesenchymal transition

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

PubMed

Tansriratanawong, Kallapat; Ishikawa, Hiroshi; Toyomura, Junko; Sato, Soh

2017-10-01

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice.

PubMed

He, Lan; Law, Priscilla T Y; Boon, Siaw Shi; Zhang, Chuqing; Ho, Wendy C S; Banks, Lawrence; Wong, C K; Chan, Juliana C N; Chan, Paul K S

2016-01-01

Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed in-vivo study of interaction between HPV and co-factors of tumorigenesis in syngeneic mice. Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment.

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells.

PubMed

Sessa, F; Bonato, M; Frigerio, B; Capella, C; Solcia, E; Prat, M; Bara, J; Samloff, I M

1990-06-01

It has been found by immunohistochemical staining that antigens normally found in gastric and/or intestinal epithelial cells are expressed in most differentiated duct cell carcinomas of the pancreas. Among 88 such tumors, 93% and 92%, respectively, expressed M1 and cathepsin E, markers of gastric surface-foveolar epithelial cells, 51% expressed pepsinogen II, a marker of gastroduodenal mucopeptic cells, 48% expressed CAR-5, a marker of colorectal epithelial cells, and 35% expressed M3SI, a marker of small intestinal goblet cells. Most of the tumors also expressed normal pancreatic duct antigens; 97% expressed DU-PAN-2, and 59% expressed N-terminus gastrin-releasing peptide. In agreement with these findings, electron microscopy revealed malignant cells with fine structural features of gastric foveolar cells, gastric mucopeptic cells, intestinal goblet cells, intestinal columnar cells, pancreatic duct epithelial cells, and cells with features of more than one cell type. Normal pancreatic duct epithelium did not express any marker of gastrointestinal epithelial cells, whereas such benign lesions as mucinous cell hypertrophy and papillary hyperplasia commonly expressed gut-type antigens but rarely expressed pancreatic duct cell markers. By contrast, lesions characterized by atypical papillary hyperplasia commonly expressed both gastric and pancreatic duct cell markers. Metaplastic pyloric-type glands expressed pepsinogen II and, except for their expression of cathepsin E, were indistinguishable from normal pyloric glands. In marked contrast, the immunohistochemical and ultrastructural features of 14 ductuloacinar cell tumors were those of cells lining terminal ductules, centroacinar cells, and/or acinar cells; none expressed any gut-type antigen. The results indicate that gastrointestinal differentiation is common in both benign and malignant lesions of pancreatic duct epithelium and suggest that duct cell carcinomas are histogenetically related to gastric- and intestinal-type metaplastic changes of epithelial cells lining the main and interlobular ducts of the pancreas.

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

PubMed

Yamamoto, Naoki; Kato, Yoshinao; Sato, Atsushi; Hiramatsu, Noriko; Yamashita, Hiromi; Ohkuma, Mahito; Miyachi, Ei-Ichi; Horiguchi, Masayuki; Hirano, Koji; Kojima, Hajime

2016-08-01

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines.

PubMed

Peel, D J; Johnson, S A; Milner, M J

1990-01-01

We have examined the ultrastructure of cellular vesicles in primary cultures of wing imaginal disc cells of Drosophila melanogaster. These cells maintain the apico-basal polarity characteristic of epithelial cells. The apical surfaces secrete extracellular material into the lumen of the vesicle from plasma membrane plaques at the tip of microvilli. During the course of one passage, cells from the established cell lines grow to confluence and then aggregate into discrete condensations joined by aligned bridges of cells. Cells in these aggregates are tightly packed, and there appears to be a loss of the epithelial polarity characteristic of the vesicle cells. Elongated cell extensions containing numerous microtubules are found in aggregates, and we suggest that these may be epithelial feet involved in the aggregation process. Virus particles are commonly found both within the nucleus and the cytoplasm of cells in the aggregates.

CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miura, Yuka; Hagiwara, Natsumi; Radisky, Derek C.

2014-09-10

Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells.more » Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.« less

Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport.

PubMed

Veszelka, Szilvia; Tóth, András; Walter, Fruzsina R; Tóth, Andrea E; Gróf, Ilona; Mészáros, Mária; Bocsik, Alexandra; Hellinger, Éva; Vastag, Monika; Rákhely, Gábor; Deli, Mária A

2018-01-01

Cell culture-based blood-brain barrier (BBB) models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC), ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA). As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L), and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1) and influx transporters (GLUT-1, LAT-1) were present in all models at mRNA levels. The transcript of BCRP (ABCG2) was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which are substrates of these transporters. Brain endothelial cell lines GP8, RBE4, D3 and D3L did not form a restrictive paracellular barrier necessary for screening small molecular weight pharmacons. Therefore, among the tested culture models, the primary cell-based EPA model is suitable for the functional analysis of the BBB.

A Comprehensive Panel of Three-Dimensional Models for Studies of Prostate Cancer Growth, Invasion and Drug Responses

PubMed Central

Härmä, Ville; Virtanen, Johannes; Mäkelä, Rami; Happonen, Antti; Mpindi, John-Patrick; Knuuttila, Matias; Kohonen, Pekka; Lötjönen, Jyrki; Kallioniemi, Olli; Nees, Matthias

2010-01-01

Prostate epithelial cells from both normal and cancer tissues, grown in three-dimensional (3D) culture as spheroids, represent promising in vitro models for the study of normal and cancer-relevant patterns of epithelial differentiation. We have developed the most comprehensive panel of miniaturized prostate cell culture models in 3D to date (n = 29), including many non-transformed and most currently available classic prostate cancer (PrCa) cell lines. The purpose of this study was to analyze morphogenetic properties of PrCa models in 3D, to compare phenotypes, gene expression and metabolism between 2D and 3D cultures, and to evaluate their relevance for pre-clinical drug discovery, disease modeling and basic research. Primary and non-transformed prostate epithelial cells, but also several PrCa lines, formed well-differentiated round spheroids. These showed strong cell-cell contacts, epithelial polarization, a hollow lumen and were covered by a complete basal lamina (BL). Most PrCa lines, however, formed large, poorly differentiated spheroids, or aggressively invading structures. In PC-3 and PC-3M cells, well-differentiated spheroids formed, which were then spontaneously transformed into highly invasive cells. These cell lines may have previously undergone an epithelial-to-mesenchymal transition (EMT), which is temporarily suppressed in favor of epithelial maturation by signals from the extracellular matrix (ECM). The induction of lipid and steroid metabolism, epigenetic reprogramming, and ECM remodeling represents a general adaptation to 3D culture, regardless of transformation and phenotype. In contrast, PI3-Kinase, AKT, STAT/interferon and integrin signaling pathways were particularly activated in invasive cells. Specific small molecule inhibitors targeted against PI3-Kinase blocked invasive cell growth more effectively in 3D than in 2D monolayer culture, or the growth of normal cells. Our panel of cell models, spanning a wide spectrum of phenotypic plasticity, supports the investigation of different modes of cell migration and tumor morphologies, and will be useful for predictive testing of anti-cancer and anti-metastatic compounds. PMID:20454659

Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

PubMed

Choo, C K; Ling, M T; Chan, K W; Tsao, S W; Zheng, Z; Zhang, D; Chan, L C; Wong, Y C

1999-08-01

The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression. Copyright 1999 Wiley-Liss, Inc.

Differences in Chlamydia trachomatis Serovar E Growth Rate in Polarized Endometrial and Endocervical Epithelial Cells Grown in Three-Dimensional Culture▿

PubMed Central

Guseva, Natalia V.; Dessus-Babus, Sophie; Moore, Cheryl G.; Whittimore, Judy D.; Wyrick, Priscilla B.

2007-01-01

In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells. PMID:17088348

Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.

Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomousmore » growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a valuable model for arsenic-induced lung cancer.« less

Flat Epithelial Atypia of the Breast.

PubMed

Collins, Laura C

2009-06-01

Lesions of the breast characterized by enlarged terminal duct lobular units lined by columnar epithelial cells are being encountered increasingly in breast biopsy specimens. Some of these lesions feature cuboidal to columnar epithelial cells in which the lining cells exhibit cytologic atypia. The role of these lesions (recently designated "flat epithelial atypia" [FEA]) in breast tumor progression is still emerging. FEA commonly coexists with well-developed examples of atypical ductal hyperplasia, low-grade ductal carcinoma in situ, lobular neoplasia, and tubular carcinoma. These findings and those of recent genetic studies suggest that FEA is a neoplastic lesion that may represent a precursor to or the earliest morphologic manifestation of ductal carcinoma in situ. Additional studies are needed to better understand the biologic nature and clinical significance of these lesions. Copyright © 2009 Elsevier Inc. All rights reserved.

Candida albicans triggers interleukin-8 secretion by oral epithelial cells.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-04-01

Oropharyngeal candidiasis is a frequent opportunistic infection associated with immunocompromised hosts. Candida albicans is the principal species responsible for this infection. Production of interleukin-8 (IL-8), by oral epithelial cells can be expected to play a major role in the recruitment and activation of professional phagocytes at the infected site. The purpose of this study was to determine whether C. albicans triggers secretion of IL-8 by oral epithelial cells in vitro and investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines (SCC4, SCC15, and OKF6/TERT-2) as well as primary gingival epithelial cells were used. Epithelial cells were cocultured with C. albicans, strains SC5314, ATCC28366 or ATCC32077, for 24-48 hr, and supernatants were analyzed for IL-8 content by ELISA. A germination-deficient mutant (efg1/efg1 cph1/cph1), otherwise isogenic to strain SC5314, was used to assess the requirement for germination in triggering IL-8 responses. In order to ascertain whether direct contact of yeast with host cells is required to trigger cytokine production, epithelial cells were separated from yeast using cell culture inserts. To test whether IL-8 secretion is dependent on IL-1alpha activity, epithelial cells were challenged with viable C. albicans in the presence or absence of neutralizing anti-IL-1alpha antibody or IL-1ra, and IL-8 secretion was measured in the supernatants. All cell lines and primary cultures responded to C. albicans with an increase in IL-8 secretion. IL-8 responses were contact-dependent, strain-specific, required yeast viability and germination into hyphae, and were in part autoregulated by IL-1alpha. Copyright 2003 Elsevier Science Ltd.

Granulocyte-macrophage colony-stimulating factor responses of oral epithelial cells to Candida albicans.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-06-01

Candida albicans is the principal fungal species responsible for oropharyngeal candidiasis, the most frequent opportunistic infection associated with immune deficiencies. Cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), are important in the generation of effective immunity to C. albicans. The purposes of this investigation were to determine whether C. albicans triggers secretion of GM-CSF by oral epithelial cells in vitro and to investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines as well as primary oral mucosal epithelial cells were challenged with stationary phase viable C. albicans, added to human cell cultures at varying yeast:oral cell ratios. Yeast were allowed to germinate for up to 48 h and supernatants were analyzed for GM-CSF by ELISA. Fixed organisms, germination-deficient mutants and separation of yeast from epithelial cells using cell culture inserts were used to assess the effects of viability, germination and physical contact, respectively, on the GM-CSF responses of these cells. Two out of three cell lines and three out of six primary cultures responded to C. albicans with an increase in GM-CSF secretion. GM-CSF responses were contact-dependent, strain-dependent, required yeast viability and were optimal when the yeast germinated into hyphae.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vilches, J.; Lopez, A.; Martinez, M.C.

This paper discusses the value of scanning electron microscopy (SEM) and x-ray microanalysis in the classification of craniopharyngiomas. This neoplasm shows epithelial nest, cords of cuboid cells, foci of squamous metaplasia, and microcystic degeneration. SEM reveals that the epithelial cysts are lined with elongated cells that possess numerous microvilli and blebs and that some cysts are lined with polyhedral cells. The microvilli are interpreted as characteristic of the fast growing craniopharyngiomas. A microanalytical study of the calcified areas reveals the presence of magnesium, phosphorus, and calcium.

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

PubMed Central

Vooijs, W. C.; Otten, H. G.; van Vliet, M.; van Dijk, A. J.; de Weger, R. A.; de Boer, M.; Bohlen, H.; Bolognesi, A.; Polito, L.; de Gast, G. C.

1997-01-01

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration. Images Figure 1 PMID:9365164

Photodynamic treatment (PDT) of endometrium primary cultures serving as an in-vitro-model for endometriosis

NASA Astrophysics Data System (ADS)

Werter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

Photodynamic therapy (PDT) of endometrium primary cultures serving as an in-vitro model for endometriosis

NASA Astrophysics Data System (ADS)

Herter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

CDKL2 promotes epithelial-mesenchymal transition and breast cancer progression

PubMed Central

Li, Linna; Liu, Chunping; Amato, Robert J.; Chang, Jeffrey T.; Du, Guangwei; Li, Wenliang

2014-01-01

The epithelial–mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by epithelial cancer cells. To identify novel regulators of EMT, we carried out cDNA screens that covered 500 human kinases. Subsequent characterization of candidate kinases led us to uncover cyclin-dependent kinase-like 2 (CDKL2) as a novel potent promoter for EMT and breast cancer progression. CDKL2-expressing human mammary gland epithelial cells displayed enhanced mesenchymal traits and stem cell-like phenotypes, which was acquired through activating a ZEB1/E-cadherin/β-catenin positive feedback loop and regulating CD44 mRNA alternative splicing to promote conversion of CD24high cells to CD44high cells. Furthermore, CDKL2 enhanced primary tumor formation and metastasis in a breast cancer xenograft model. Notably, CDKL2 is expressed significantly higher in mesenchymal human breast cancer cell lines than in epithelial lines, and its over-expression/amplification in human breast cancers is associated with shorter disease-free survival. Taken together, our study uncovered a major role for CDKL2 in promoting EMT and breast cancer progression. PMID:25333262

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

PubMed

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

2014-01-01

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

CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation

PubMed Central

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

2014-01-01

Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs). In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF) = 1.3), and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs) with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients. PMID:25536195

Establishment and characterization of immortalized gingival epithelial and fibroblastic cell lines for the development of organotypic cultures.

PubMed

Bao, Kai; Akguel, Baki; Bostanci, Nagihan

2014-01-01

In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro. © 2014 S. Karger AG, Basel.

Cell reintegration: Stray epithelial cells make their way home.

PubMed

Wilson, Tyler J; Bergstralh, Dan T

2017-06-01

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

Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

PubMed

Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

2017-10-01

The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Early Molecular Events in Murine Gastric Epithelial Cells Mediated by Helicobacter pylori CagA.

PubMed

Banerjee, Aditi; Basu, Malini; Blanchard, Thomas G; Chintalacharuvu, Subba R; Guang, Wei; Lillehoj, Erik P; Czinn, Steven J

2016-10-01

Murine models of Helicobacter pylori infection are used to study host-pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events. © 2016 John Wiley & Sons Ltd.

Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

PubMed

Fiscus, R R; Leung, C P; Yuen, J P; Chan, H C

2001-01-01

Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines. Copyright 2001 Academic Press.

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

Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.

PubMed

Riches, Andrew; Campbell, Elaine; Borger, Eva; Powis, Simon

2014-03-01

Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined. We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes. Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control. These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

Establishment and characterization of a telomerase immortalized human gingival epithelial cell line.

PubMed

Moffatt-Jauregui, C E; Robinson, B; de Moya, A V; Brockman, R D; Roman, A V; Cash, M N; Culp, D J; Lamont, R J

2013-12-01

Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase immortalized human gingival epithelial cell line and compare its in vitro behaviour to that of human GECs. Human primary GECs were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, telomerase immortalized gingival keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors and invasion by P. gingivalis. TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for toll-like receptors 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild-type P. gingivalis and an invasion defective ΔserB mutant. Results confirm bmi1/hTERT immortalization of primary GECs generated a robust cell line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development of a chick embryo heart cell for the cultivation of poliovirus.

PubMed

PRIER, J E; SULLIVAN, R

1959-04-17

An epithelial-like cell has been developed in line culture that apparently is stable. Although initially isolated cells were incapable of supporting the growth of poliovirus, the cells of the sixth and later passages allowed virus to propagate. The early, nonsusceptible cells were fibroblastic in appearance, in contrast to the epithelial type, poliovirussusceptible, derived cell of later passages.

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

PubMed

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

2014-01-01

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

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

NASA Astrophysics Data System (ADS)

Armitage, Mark

Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or mutant conformation in all the above cells lines and two other control lines HOS (a human osteosarcoma cell line) and H Tori-3 (SV40 immortalised thyroid epithelial cells).

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties. | Office of Cancer Genomics

Cancer.gov

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines.

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells.

PubMed

Yang, Min; Ma, Bo; Shao, Hanshuang; Clark, Amanda M; Wells, Alan

2016-07-07

Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due to malignant metastases in breast cancer.

The therapeutic effects of docosahexaenoic acid on oestrogen/androgen-induced benign prostatic hyperplasia in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chao; Luo, Fei; Zhou, Ying

Benign prostatic hyperplasia (BPH) is one of the major disorders of the urinary system in elderly men. Docosahexaenoic acid (DHA) is the main component of n-3 polyunsaturated fatty acids (n-3 PUFAs) and has nerve protective, anti-inflammatory and tumour-growth inhibitory effects. Here, the therapeutic potential of DHA in treating BPH was investigated. Seal oil effectively prevented the development of prostatic hyperplasia induced by oestradiol/testosterone in a rat model by suppressing the increase of the prostatic index (PI), reducing the thickness of the peri-glandular smooth muscle layer, inhibiting the proliferation of both prostate epithelial and stromal cells, and downregulating the expression ofmore » androgen receptor (AR) and oestrogen receptor α (ERα). An in vitro study showed that DHA inhibited the growth of the human prostate stromal cell line WPMY-1 and the epithelial cell line RWPE-1 in a dose- and time-dependent manner. In both cell lines, the DHA arrested the cell cycle in the G2/M phase. In addition, DHA also reduced the expression of ERα and AR in the WPMY-1 and RWPE-1 cells. These results indicate that DHA inhibits the multiplication of prostate stromal and epithelial cells through a mechanism that may involve cell cycle arrest and the downregulation of ERα and AR expression. - Highlights: • Seal oil prevents oestradiol/testosterone (E2/T)-induced BPH in castrated rats. • Seal oil downregulates the expression of oestrogen receptor α(ERα) and androgen receptor (AR) in rat BPH tissues. • DHA inhibits the growth of human prostate stromal and epithelial cells in vitro. • DHA arrests human prostate stromal and epithelial cells in the G2/M phase and downregulates the expression of cyclin B1. • DHA inhibits the expression of ERα and AR in human prostate stromal and epithelial cells.« less

The Pseudomonas aeruginosa exopolysaccharide Psl facilitates surface adherence and NF-kappaB activation in A549 cells.

PubMed

Byrd, Matthew S; Pang, Bing; Mishra, Meenu; Swords, W Edward; Wozniak, Daniel J

2010-06-29

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213-8221, 2006). Using an NF-kappaB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-kappaB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-kappaB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-kappaB activation, likely as a result of increasing contact between bacterial cells and epithelial cells.

The Pseudomonas aeruginosa Exopolysaccharide Psl Facilitates Surface Adherence and NF-κB Activation in A549 Cells

PubMed Central

Byrd, Matthew S.; Pang, Bing; Mishra, Meenu; Swords, W. Edward; Wozniak, Daniel J.

2010-01-01

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213–8221, 2006). Using an NF-κB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-κB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-κB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-κB activation, likely as a result of increasing contact between bacterial cells and epithelial cells. PMID:20802825

Endoplasmic reticulum chaperone GRP78 mediates cigarette smoke-induced necroptosis and injury in bronchial epithelium.

PubMed

Wang, Yong; Zhou, Jie-Sen; Xu, Xu-Chen; Li, Zhou-Yang; Chen, Hai-Pin; Ying, Song-Min; Li, Wen; Shen, Hua-Hao; Chen, Zhi-Hua

2018-01-01

Bronchial epithelial cell death and airway inflammation induced by cigarette smoke (CS) have been involved in the pathogenesis of COPD. GRP78, belonging to heat shock protein 70 family, has been implicated in cell death and inflammation, while little is known about its roles in COPD. Here, we demonstrate that GRP78 regulates CS-induced necroptosis and injury in bronchial epithelial cells. GRP78 and necroptosis markers were examined in human bronchial epithelial (HBE) cell line, primary mouse tracheal epithelial cells, and mouse lungs. siRNA targeting GRP78 gene and necroptosis inhibitor were used. Expression of inflammatory cytokines, mucin MUC5AC, and related signaling pathways were detected. Exposure to CS significantly increased the expression of GRP78 and necroptosis markers in HBE cell line, primary mouse tracheal epithelial cells, and mouse lungs. Inhibition of GRP78 significantly suppressed CS extract (CSE)-induced necroptosis. Furthermore, GRP78-necroptosis cooperatively regulated CSE-induced inflammatory cytokines such as interleukin 6 (IL6), IL8, and mucin MUC5AC in HBE cells, likely through the activation of nuclear factor (NF-κB) and activator protein 1 (AP-1) pathways, respectively. Taken together, our results demonstrate that GRP78 promotes CSE-induced inflammatory response and mucus hyperproduction in airway epithelial cells, likely through upregulation of necroptosis and subsequent activation of NF-κB and AP-1 pathways. Thus, inhibition of GRP78 and/or inhibition of necroptosis could be the effective therapeutic approaches for the treatment of COPD.

Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

PubMed

Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

2015-09-01

Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. Copyright © 2015. Published by Elsevier B.V.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Shan; Wang, Ming-Wei; Yao, Xiaoqiang

2009-05-15

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newlymore » developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.« less

Thalidomide distinctly affected TNF-α, IL-6 and MMP secretion by an ovarian cancer cell line (SKOV-3) and primary ovarian cancer cells.

PubMed

Piura, Benjamin; Medina, Liat; Rabinovich, Alex; Dyomin, Victor; Huleihel, Mahmoud

2013-01-01

Thalidomide inhibits TNF-α production in lipopolysaccharide-stimulated monocytes. The aim of this study was to evaluate the effect of thalidomide on TNF-α, IL-6 and MMP secretion in epithelial ovarian carcinoma cells. SKOV-3 cells and primary epithelial ovarian carcinoma cells were cultured in the presence of various concentrations of thalidomide. Cell proliferation was examined by MTT proliferation assay. TNF-α and IL-6 levels were determined in the supernatants of the cell cultures by ELISA, and MMP activity was examined by gelatin zymography. Thalidomide did not significantly affect the proliferation and growth of SKOV-3 cells. However, it decreased significantly the capacity of SKOV-3 cells and primary epithelial ovarian carcinoma cells to secrete TNF-α. Thalidomide also significantly decreased the capacity of SKOV-3 cells, but not primary epithelial ovarian carcinoma cells, to secrete MMP-9 and MMP-2. However, thalidomide did not affect IL-6 secretion in SKOV-3 cells or primary epithelial ovarian carcinoma cells. Our study suggests that thalidomide distinctly affected TNF-α, IL-6 and MMPs secretion by an ovarian carcinoma cell line (SKOV-3) and primary ovarian cancer cells. This might suggest a different susceptibility of these two types of cells to thalidomide, and/or that the mechanisms of secretion of the factors examined are differently regulated in these cells. Our results may deepen our understanding the mechanism/s of action of thalidomide in ovarian carcinoma cells. The results might have important implications in future therapeutic strategies that will incorporate thalidomide and other cytokine inhibitors in the treatment of epithelial ovarian carcinoma.

The world of epithelial sheets.

PubMed

Honda, Hisao

2017-06-01

An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

EphA3 maintains radioresistance in head and neck cancers through epithelial mesenchymal transition.

PubMed

Kim, Song Hee; Lee, Won Hyeok; Kim, Seong Who; Je, Hyoung Uk; Lee, Jong Cheol; Chang, Hyo Won; Kim, Young Min; Kim, Kyungbin; Kim, Sang Yoon; Han, Myung Woul

2018-07-01

Radiotherapy is a well-established therapeutic modality used in the treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after radiation treatment. Thus, many studies have attempted to identify effective radiosensitizers. Eph receptor functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains unclear. In the current study, we established a stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and found that EphA3 was expressed predominantly in the radioresistant head and neck cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we found that EphA3 was overexpressed in recurrent laryngeal cancer specimens after radiation therapy. EphA3 mediated the tumor invasiveness and migration in radioresistant head and neck cancer cell lines and epithelial mesenchymal transition- related protein expression. Inhibition of EphA3 enhanced radiosensitivity in the AMC HN 3R cell line in vitro and in vivo study. In conclusion, our results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and plays a crucial role in the development of radioresistance in head and neck cancers by regulating the epithelial mesenchymal transition pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Immunohistochemical evaluation of inducible nitric oxide synthase in the epithelial lining of odontogenic cysts: A qualitative and quantitative analysis.

PubMed

Akshatha, B K; Karuppiah, Karpagaselvi; Manjunath, G S; Kumarswamy, Jayalakshmi; Papaiah, Lokesh; Rao, Jyothi

2017-01-01

The three common odontogenic cysts include radicular cysts (RCs), dentigerous cysts (DCs), and odontogenic keratocysts (OKCs). Among these 3 cysts, OKC is recently been classified as benign keratocystic odontogenic tumor attributing to its aggressive behavior, recurrence rate, and malignant potential. The present study involved qualitative and quantitative analysis of inducible nitric oxide synthase (iNOS) expression in epithelial lining of RCs, DCs, and OKCs, compare iNOS expression in epithelial linings of all the 3 cysts and determined overexpression of iNOS in OKCs which might contribute to its aggressive behavior and malignant potential. The present study is to investigate the role of iNOS in the pathogenesis of OKCs, DCs, and RCs by evaluating the iNOS expression in the epithelial lining of these cysts. Analysis of iNOS expression in epithelial lining cells of 20 RCs, 20 DCs, and 20 OKCs using immunohistochemistry done. The percentage of positive cells and intensity of stain was assessed and compared among all the 3 cysts using contingency coefficient. Kappa statistics for the two observers were computed for finding interobserver agreement. The percentage of iNOS-positive cells was found to be remarkably high in OKCs (12/20) -57.1% as compared to RCs (6/20) - 28.6% and DCs (3/20) - 14.3%. The interobserver agreement for iNOS-positive percentage cells was arrived with kappa values with OKCs → Statistically significant ( P > 0.000), RCs → statistically significant ( P > 0.001) with no significant values for DCs. No statistical difference exists among 3 study samples in regard to the intensity of staining with iNOS. Increased iNOS expression in OKCs may contribute to bone resorption and accumulation of wild-type p53, hence, making OKCs more aggressive.

A Telomerase Immortalized Human Proximal Tubule Cell Line with a Truncation Mutation (Q4004X) in Polycystin-1

PubMed Central

Herbert, Brittney-Shea; Grimes, Brenda R.; Xu, Wei Min; Werner, Michael; Ward, Christopher; Rossetti, Sandro; Harris, Peter; Bello-Reuss, Elsa; Ward, Heather H.; Miller, Caroline; Gattone, Vincent H.; Phillips, Carrie L.; Wandinger-Ness, Angela; Bacallao, Robert L.

2013-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions [1], [2]. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X) in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells. PMID:23383103

Cystic fibrosis epithelial cells are primed for apoptosis as a result of increased Fas (CD95).

PubMed

Chen, Qiwei; Pandi, Sudha Priya Soundara; Kerrigan, Lauren; McElvaney, Noel G; Greene, Catherine M; Elborn, J Stuart; Taggart, Clifford C; Weldon, Sinéad

2018-02-24

Previous work suggests that apoptosis is dysfunctional in cystic fibrosis (CF) airways with conflicting results. We evaluated the relationship between dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and apoptosis in CF airway epithelial cells. Apoptosis and associated caspase activity were analysed in non-CF and CF tracheal and bronchial epithelial cell lines. Basal levels of apoptosis and activity of caspase-3 and caspase-8 were significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells and bronchial brushings from CF patients compared to non-CF controls. Neutralisation of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. In addition, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway, which may be associated with dysfunctional CFTR. Copyright © 2018 European Cystic Fibrosis Society. All rights reserved.

NANOG regulates epithelial-mesenchymal transition and chemoresistance in ovarian cancer.

PubMed

Qin, Shan; Li, Yanfang; Cao, Xuexia; Du, Jiexian; Huang, Xianghua

2017-02-28

A key transcription factor associated with poor prognosis and resistance to chemotherapy in ovarian cancer is NANOG. However, the mechanism by which NANOG functions remains undefined. It has been suggested that epithelial-to-mesenchymal transition (EMT) also contributes to development of drug resistance in different cancers. We thus determined whether NANOG expression was associated with EMT and chemoresistance in epithelial ovarian cancer cells. NANOG expression was increased in epithelial ovarian cancer cell lines compared with its expression in normal epithelial ovarian cell lines. NANOG expression in SKOV-3 or OV2008 cells directly correlated with high expression of mesenchymal cell markers and inversely with low expression of epithelial cell marker. RNAi-mediated silencing of NANOG in SKOV-3 reversed the expression of mesenchymal cell markers and restored expression of E-cadherin. Reversibly, stable overexpression of NANOG in Moody cells increased expression of N-cadherin whereas down-regulating expression of E-cadherin, cumulatively indicating that NANOG plays an important role in maintaining the mesenchymal cell markers. Modulating NANOG expression did not have any effect on proliferation or colony formation. Susceptibility to cisplatin increased in SKOV-3 cells on down-regulating NANOG and reversible results were obtained in Moody cells post-overexpression of NANOG. NANOG silencing in SKOV-3 and OV2008 robustly attenuated in vitro migration and invasion. NANOG expression exhibited a biphasic pattern in patients with ovarian cancer and expression was directly correlated to chemoresistance retrospectively. Cumulatively, our data demonstrate that NANOG expression modulates chemosensitivity and EMT resistance in ovarian cancer. © 2017 The Author(s).

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

PubMed

Fernando, Elizabeth H; Dicay, Michael; Stahl, Martin; Gordon, Marilyn H; Vegso, Andrew; Baggio, Cristiane; Alston, Laurie; Lopes, Fernando; Baker, Kristi; Hirota, Simon; McKay, Derek M; Vallance, Bruce; MacNaughton, Wallace K

2017-11-01

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner. Copyright © 2017 the American Physiological Society.

Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

PubMed

Meir, Michael; Flemming, Sven; Burkard, Natalie; Bergauer, Lisa; Metzger, Marco; Germer, Christoph-Thomas; Schlegel, Nicolas

2015-10-15

Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling. Copyright © 2015 the American Physiological Society.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Feng; Jordan, Ashley; Kluz, Thomas

The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealedmore » the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.« less

Cell death at the intestinal epithelial front line.

PubMed

Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

2016-07-01

The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells.

PubMed

Jiang, Feng; Saunders, Beatriz O; Haller, Edward; Livingston, Sandra; Nicosia, Santo V; Bai, Wenlong

2003-01-01

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

PubMed

Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

2017-08-01

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Contribution of the Airway Epithelial Cell to Host Defense.

PubMed

Stanke, Frauke

2015-01-01

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

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Hongzhen; Zhou Jianjun; Miki, Jun

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

PubMed Central

Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

2009-01-01

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO METALS

EPA Science Inventory

We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms respons...

The role of IL-1beta in reduced IL-7 production by stromal and epithelial cells: a model for impaired T-cell numbers in the gut during HIV-1 infection.

PubMed

Thang, P H; Ruffin, N; Brodin, D; Rethi, B; Cam, P D; Hien, N T; Lopalco, L; Vivar, N; Chiodi, F

2010-08-01

Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production. We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods. Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

Carbapenem resistance confers to Klebsiella pneumoniae strains an enhanced ability to induce infection and cell death in epithelial tissue-specific in vitro models.

PubMed

Leone, Laura; Raffa, Salvatore; Martinelli, Daniela; Torrisi, Maria Rosaria; Santino, Iolanda

2015-01-01

Carbapenem-resistant Klebsiella pneumoniae strains (KPC-Kp) are emerging worldwide causing different nosocomial infections including those of the urinary tract, lung or skin wounds. For these strains, the antibiotic treatment is limited to only few choices including colistin, whose continuous use led to the emergence of carbapenem-resistant KPC-Kp strains resistant also to this treatment (KPC-Kp Col-R). Very little is known about the capacity of the different strains of KPC-Kp to invade the epithelial cells in vitro. To verify if the acquisition of carbapenem-resistant and the colistin-resistant phenotypes are correlated with a different ability to infect a series of epithelial cell lines of various tissutal origin and with a different capacity to induce cellular death. We used Klebsiella pneumoniae (KP), KPC-Kp and KPC-Kp Col-R strains, isolated from different patients carrying various tissue-specific infections, to infect a series of epithelial cell lines of different tissutal origin. The invasive capacity of the strains and the extent and characteristics of the cell damage and death induced by the bacteria were evaluated and compared. Our results show that both KPC-Kp and KPC-Kp Col-R display a greater ability to infect the epithelial cells, with respect to KP, and that the bacterial cell invasion results in a nonprogrammed cell death.

Different tocopherol isoforms vary in capacity to scavenge free radicals, prevent inflammatory response, and induce apoptosis in both adult- and fetal-derived intestinal epithelial cells.

PubMed

Elisia, Ingrid; Kitts, David D

2013-01-01

Gamma-tocopherol (γ-Toc) and δ-Toc are two vitamin E isoforms for which biological activities are not well established, yet these isoforms are present in many different sources of vegetable oils and, therefore, contribute significantly to the total dietary intake of vitamin E. Infant formula also contains relatively high amounts of γ-Toc and δ-Toc, compared with that found in human milk. The efficacy of γ-Toc and δ-Toc to modulate cellular events that include oxidative stress, inflammatory response, and apoptosis-mediated cytotoxicity, relative to α-Toc, was determined using differentiated Caco-2 and primary FHs 74 Int cells intestinal epithelial cell lines. Antioxidant capacity of Toc-isoforms followed the order of δ-Toc > γ-Toc > α-Toc against peroxyl radical-induced membrane oxidation in both Caco-2 and FHs 74 Int cells, respectively. The different Toc-isoforms suppressed inflammatory response in interferon (IFN) γ/phorbol myristate acetate (PMA)-induced Caco-2 adult-derived intestinal epithelial cells, but exacerbated both IL8 and PGE2 secretion in fetal-derived FHs 74 Int intestinal epithelial cells. Lastly, Toc exhibited an isoform-dependent apoptosis-mediated cytotoxicity, whereby δ-Toc elicited the greatest apoptosis followed by γ-Toc, whereas α-Toc was not cytotoxic. Cellular uptake of non-α-Toc isoforms were greater (P < 0.05) than that observed for α-Toc in both intestinal epithelial cell lines which in part explains the superior bioactive function observed for both γ-Toc and δ-Toc, compared with α-Toc. We conclude that the non-α-Toc isoforms of vitamin E have distinct roles that influence oxidative stress and inflammatory responses in both adult and fetal-derived intestinal epithelial cell lines. © 2013 International Union of Biochemistry and Molecular Biology.

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

PubMed

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

2014-01-01

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

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

PubMed Central

Zhou, Jian; Alvarez-Elizondo, Martha B.; Botvinick, Elliot

2012-01-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca2+ wave in the epithelium, and multiple Ca2+ waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca2+ or decreasing intracellular Ca2+ both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca2+-dependent smooth muscle shortening. PMID:22114176

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.

PubMed

Zhou, Jian; Alvarez-Elizondo, Martha B; Botvinick, Elliot; George, Steven C

2012-02-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

Epithelial cell specific properties and genetic complementation in a delta F508 cystic fibrosis nasal polyp cell line.

PubMed

Kunzelmann, K; Lei, D C; Eng, K; Escobar, L C; Koslowsky, T; Gruenert, D C

1995-09-01

Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, delta F508 homozygote, cell line that has tight junctions pre-crisis. The cells (sigma CFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl- transport as measured by efflux of radioactive Cl-, short circuit current (Isc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K(+)- and Ca(+2)-dependent Cl- transport. Measurement of 36Cl- efflux following stimulation with A23187 and ionomycin indicate a Ca(+2)-dependent Cl- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 degrees C for 48 h there is no indication of cAMP-dependent Cl- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells.(ABSTRACT TRUNCATED AT 250 WORDS)

β2 adrenergic agonist suppresses eosinophil-induced epithelial-to-mesenchymal transition of bronchial epithelial cells.

PubMed

Kainuma, Keigo; Kobayashi, Tetsu; D'Alessandro-Gabazza, Corina N; Toda, Masaaki; Yasuma, Taro; Nishihama, Kota; Fujimoto, Hajime; Kuwabara, Yu; Hosoki, Koa; Nagao, Mizuho; Fujisawa, Takao; Gabazza, Esteban C

2017-05-02

Epithelial-mesenchymal transition is currently recognized as an important mechanism for the increased number of myofibroblasts in cancer and fibrotic diseases. We have already reported that epithelial-mesenchymal transition is involved in airway remodeling induced by eosinophils. Procaterol is a selective and full β 2 adrenergic agonist that is used as a rescue of asthmatic attack inhaler form and orally as a controller. In this study, we evaluated whether procaterol can suppress epithelial-mesenchymal transition of airway epithelial cells induced by eosinophils. Epithelial-mesenchymal transition was assessed using a co-culture system of human bronchial epithelial cells and primary human eosinophils or an eosinophilic leukemia cell line. Procaterol significantly inhibited co-culture associated morphological changes of bronchial epithelial cells, decreased the expression of vimentin, and increased the expression of E-cadherin compared to control. Butoxamine, a specific β 2 -adrenergic antagonist, significantly blocked changes induced by procaterol. In addition, procaterol inhibited the expression of adhesion molecules induced during the interaction between eosinophils and bronchial epithelial cells, suggesting the involvement of adhesion molecules in the process of epithelial-mesenchymal transition. Forskolin, a cyclic adenosine monophosphate-promoting agent, exhibits similar inhibitory activity of procaterol. Overall, these observations support the beneficial effect of procaterol on airway remodeling frequently associated with chronic obstructive pulmonary diseases.

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

PubMed

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

2005-10-01

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

Role of medullary progenitor cells in epithelial cell migration and proliferation

PubMed Central

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

2014-01-01

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

The Impact of Ethnicity-Dependent Differences in Breast Epithelial Hierarchy on Tumor Incidence and Characteristics

DTIC Science & Technology

2016-10-01

TNBC) is significantly higher in African American than Caucasian women suggesting that the biology of normal breast epithelial cells between these two...we have generated immortalized cell lines from healthy breast tissues of African American and Caucasian women and transformed these cells with...progenitor phenotype. Transformed cells are being characterized for signal transduction pathway activation. Transformed cells from African American women

Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation

PubMed Central

Rajavelu, Priya; Chen, Gang; Xu, Yan; Kitzmiller, Joseph A.; Korfhagen, Thomas R.; Whitsett, Jeffrey A.

2015-01-01

Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef–/– mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen. PMID:25866971

Characterization of three new serous epithelial ovarian cancer cell lines

PubMed Central

Ouellet, Véronique; Zietarska, Magdalena; Portelance, Lise; Lafontaine, Julie; Madore, Jason; Puiffe, Marie-Line; Arcand, Suzanna L; Shen, Zhen; Hébert, Josée; Tonin, Patricia N; Provencher, Diane M; Mes-Masson, Anne-Marie

2008-01-01

Background Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors (TOV-1946, and TOV-2223G), as well as the matched ascites for one case (OV-1946). Methods In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice. Results While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic TP53 mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in BRAF, KRAS or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease. Conclusion This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient. PMID:18507860

Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines.

PubMed

Guo, X; Ruiz, A; Rando, R R; Bok, D; Gudas, L J

2000-11-01

When exogenous [(3)H]retinol (vitamin A) was added to culture medium, normal human epithelial cells from the oral cavity, skin, lung and breast took up and esterified essentially all of the [(3)H]retinol within a few hours. As shown by [(3)H]retinol pulse-chase experiments, normal epithelial cells then slowly hydrolyzed the [(3)H]retinyl esters to [(3)H]retinol, some of which was then oxidized to [(3)H]retinoic acid (RA) over a period of several days. In contrast, cultured normal human fibroblasts and human umbilical vein endothelial cells (HUVEC) did not esterify significant amounts of [(3)H]retinol; this lack of [(3)H]retinol esterification was correlated with a lack of expression of lecithin:retinol acyltransferase (LRAT) transcripts in normal fibroblast and HUVEC strains. These results indicate that normal, differentiated cell types differ in their ability to esterify retinol. Human carcinoma cells (neoplastically transformed epithelial cells) of the oral cavity, skin and breast did not esterify much [(3)H]retinol and showed greatly reduced LRAT expression. Transcripts of the neutral, bile salt-independent retinyl ester hydrolase and the bile salt-dependent retinyl ester hydrolase were undetectable in all of the normal cell types, including the epithelial cells. These experiments suggest that retinoid-deficiency in the tumor cells could develop because of the lack of retinyl esters, a storage form of retinol.

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

PubMed

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

2017-12-01

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

Moderate plasma activated media suppresses proliferation and migration of MDCK epithelial cells

NASA Astrophysics Data System (ADS)

Mohades, Soheila; Laroussi, Mounir; Maruthamuthu, Venkat

2017-05-01

Low-temperature plasma has been shown to have diverse biomedical uses, including its applications in cancer and wound healing. One recent approach in treating mammalian cells with plasma is through the use of plasma activated media (PAM), which is produced by exposing cell culture media to plasma. While the adverse effects of PAM treatment on cancerous epithelial cell lines have been recently studied, much less is known about the interaction of PAM with normal epithelial cells. In this paper, non-cancerous canine kidney MDCK (Madin-Darby Canine Kidney) epithelial cells were treated by PAM and time-lapse microscopy was used to directly monitor their proliferation and random migration upon treatment. While longer durations of PAM treatment led to cell death, we found that moderate levels of PAM treatment inhibited proliferation in these epithelial cells. We also found that PAM treatment reduced random cell migration within epithelial islands. Immunofluorescence staining showed that while there were no major changes in the actin/adhesion apparatus, there was a significant change in the nuclear localization of proliferation marker Ki-67, consistent with our time-lapse results.

Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation.

PubMed

Gabastou, J M; Kernéis, S; Bernet-Camard, M F; Barbat, A; Coconnier, M H; Kaper, J B; Servin, A L

1995-09-01

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc-/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and transmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc-/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occurred in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Finite cell lines of turkey sperm storage tubule cells: ultrastructure and protein analysis

USDA-ARS?s Scientific Manuscript database

Cell lines of turkey sperm storage tubule (SST) epithelial cells were established. Turkey SSTs were dissected from freshly obtained uterovaginal junction (UVJ) tissue and placed in explants culture on various substrates and media. Primary cultures of SST epithelium only survived and grew from SST ex...

Coreceptors and Their Ligands in Epithelial γδ T Cell Biology

PubMed Central

Witherden, Deborah A.; Johnson, Margarete D.; Havran, Wendy L.

2018-01-01

Epithelial tissues line the body providing a protective barrier from the external environment. Maintenance of these epithelial barrier tissues critically relies on the presence of a functional resident T cell population. In some tissues, the resident T cell population is exclusively comprised of γδ T cells, while in others γδ T cells are found together with αβ T cells and other lymphocyte populations. Epithelial-resident γδ T cells function not only in the maintenance of the epithelium, but are also central to the repair process following damage from environmental and pathogenic insults. Key to their function is the crosstalk between γδ T cells and neighboring epithelial cells. This crosstalk relies on multiple receptor–ligand interactions through both the T cell receptor and accessory molecules leading to temporal and spatial regulation of cytokine, chemokine, growth factor, and extracellular matrix protein production. As antigens that activate epithelial γδ T cells are largely unknown and many classical costimulatory molecules and coreceptors are not used by these cells, efforts have focused on identification of novel coreceptors and ligands that mediate pivotal interactions between γδ T cells and their neighbors. In this review, we discuss recent advances in the understanding of functions for these coreceptors and their ligands in epithelial maintenance and repair processes. PMID:29686687

Mesenchymal-Epithelial Transition and Circulating Tumor Cells in Small Cell Lung Cancer.

PubMed

Hamilton, Gerhard; Rath, Barbara

2017-01-01

Cancer patients die of metastatic disease but knowledge regarding individual steps of this complex process of intravasation, spread and extravasation leading to secondary lesions is incomplete. Subpopulations of tumor cells are supposed to undergo an epithelial-mesenchymal transition (EMT), to enter the bloodstream and eventually establish metastases in a reverse process termed mesenchymal-epithelial transition (MET). Small cell lung cancer (SCLC) represents a unique model to study metastatic spread due to early dissemination and relapse, as well as availability of a panel of circulating cancer cell (CTC) lines recently. Additionally, chemosensitive SCLC tumor cells switch to a completely resistant phenotype during cancer recurrence. In advanced disease, SCLC patients display extremely high blood counts of CTCs in contrast to other tumors, like breast, prostate and colon cancer. Local inflammatory conditions at the primary tumor site and recruitment of macrophages seem to increase the shedding of tumor cells into the circulation in processes which may proceed independently of EMT. Since millions of cells are released by tumors into the circulation per day, analysis of a limited number of CTCs at specific time points are difficult to be related to the development of metastatic lesions which may occur approximately one year later. We have obtained a panel of SCLC CTC cell line from patients with relapsing disease, which share characteristic markers of this malignancy and a primarily epithelial phenotype with unique formation of large tumorospheres, containing quiescent and hypoxic cells. Although smoking and inflammation promote EMT, partial expression of vimentin indicates a transitional state with partial EMT in these cell lines at most. The CTC lines exhibit high expression of EpCAM , absent phosphorylation of β-catenin and background levels of Snail. Provided that these tumor cells had ever undergone EMT, here in advanced disease MET seem to have occurred already in the peripheral circulation. Alternative explanations for the expression of mesenchymal markers of the CTC lines are the heterogeneity of SCLC cells, cooperative migration or altered gene expression in response to the inflammatory tumor microenvironment allowing for tumor spread without EMT/MET.

Potential Role for a Carbohydrate Moiety in Anti-Candida Activity of Human Oral Epithelial Cells

PubMed Central

Steele, Chad; Leigh, Janet; Swoboda, Rolf; Ozenci, Hatice; Fidel, Paul L.

2001-01-01

Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by an as-yet-undefined carbohydrate moiety. PMID:11598085

Paratesticular cysts with benign epithelial proliferations of wolffian origin.

PubMed

Nistal, Manuel; González-Peramato, Pilar; Serrano, Alvaro; Vega-Perez, Maria; De Miguel, Maria P; Regadera, Javier

2005-08-01

Paratesticular cysts with benign epithelial proliferations (BEPs) are rare. Only 10 cases were found in a series of 431 paratesticular cysts and were classified as follows: cystadenoma, 5; papilloma, 2; and hamartoma, 3. Four cystadenomas showed multiple papillae lined by CD10+ epithelial cells with hyperchromatic nuclei. The remaining lesion showed areas with a microcystic, glandular, cribriform pattern, with small, benign glands without atypia. Urothelial papilloma presented BEPs with cytokeratin (CK) 7+ and CD10+ and CK20- umbrella-like cells. The mural papilloma was lined by proliferative cylindrical cells exhibiting strong CK7 and CD10 expression. The 3 Wolffian hamartomas were characterized by strongly CD10+ epithelium surrounded by smooth muscle cells. The consistent CD10 expression in BEPs of paratesticular cysts suggests a Wolffian origin. The differential diagnosis of paratesticular cysts with BEP vs metastatic prostatic and primary borderline or malignant tumors is discussed.

An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

PubMed

He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

2009-06-01

Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

EPA Science Inventory

We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

Luminal cholinergic signalling in airway lining fluid: a novel mechanism for activating chloride secretion via Ca2+-dependent Cl− and K+ channels

PubMed Central

Hollenhorst, Monika I; Lips, Katrin S; Wolff, Miriam; Wess, Jürgen; Gerbig, Stefanie; Takats, Zoltan; Kummer, Wolfgang; Fronius, Martin

2012-01-01

BACKGROUND AND PURPOSE Recent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this non-neuronal cholinergic system is not known in detail. Thus, this study focused on the effect of luminal ACh as a regulator of transepithelial ion transport in epithelial cells. EXPERIMENTAL APPROACH RT-PCR experiments were performed using mouse tracheal epithelial cells for ChAT and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) MS. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing-chamber experiments. KEY RESULTS Transcripts encoding ChAT and OCT1–3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose-dependent increase in the transepithelial ion current (EC50: 23.3 µM), characterized by a transient peak and sustained plateau current. This response was not affected by the Na+-channel inhibitor amiloride. The Cl−-channel inhibitor niflumic acid or the K+-channel blocker Ba2+ attenuated the ACh effect. The calcium ionophore A23187 mimicked the ACh effect. Luminal nicotine or muscarine increased the ion current. Experiments with receptor gene-deficient animals revealed the participation of muscarinic receptor subtypes M1 and M3. CONCLUSIONS AND IMPLICATIONS The presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non-neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways. PMID:22300281

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

PubMed

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Enhanced IL-1{beta}-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-{kappa}B signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muselet-Charlier, Celine; Universite Pierre et Marie Curie-Paris 6, Paris, UMR-S719, F-75012; Roque, Telma

2007-06-01

Transcription nuclear factor-{kappa}B (NF-{kappa}B) is hyperactivated in cystic fibrosis (CF) lung epithelial cells, and participates in exaggerated IL-8 production in the CF lung. We recently found that rapid activation of NF-{kappa}B occurred in a CF lung epithelial IB3-1 cell line (CF cells) upon IL-1{beta} stimulation, which was not observed in its CFTR-corrected lung epithelial S9 cell line (corrected cells). To test whether other signaling pathways such as that of mitogen-activated protein kinases (MAPKs) could be involved in IL-1{beta}-induced IL-8 production of CF cells, we investigated ERK1/2, JNK, and p38MAP signaling compared to NF-{kappa}B. Within 30 min, exposure to IL-1{beta} causedmore » high activation of NF-{kappa}B, ERK1/2, p38MAP but not JNK in CF cells compared to corrected cells. Treatment of IL-1{beta}-stimulated CF cells with a series of chemical inhibitors of NF-{kappa}B, ERK1/2, and p38MAP, when used separately, reduced slightly IL-8 production. However, when used together, these inhibitors caused a blockade in IL-1{beta}-induced IL-8 production in CF cells. Understanding of the cross-talk between NF-{kappa}B and MAPKs signaling in CF lung epithelial cells may help in developing new therapeutics to reduce lung inflammation in patients with CF.« less

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

PubMed Central

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

2014-01-01

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

Characterization of exosomes derived from ovarian cancer cells and normal ovarian epithelial cells by nanoparticle tracking analysis.

PubMed

Zhang, Wei; Peng, Peng; Kuang, Yun; Yang, Jiaxin; Cao, Dongyan; You, Yan; Shen, Keng

2016-03-01

Cellular exosomes are involved in many disease processes and have the potential to be used for diagnosis and treatment. In this study, we compared the characteristics of exosomes derived from human ovarian epithelial cells (HOSEPiC) and three epithelial ovarian cancer cell lines (OVCAR3, IGROV1, and ES-2) to investigate the differences between exosomes originating from normal and malignant cells. Two established colloid-chemical methodologies, electron microscopy (EM) and dynamic light scattering (DLS), and a relatively new method, nanoparticle tracking analysis (NTA), were used to measure the size and size distribution of exosomes. The concentration and epithelial cellular adhesion molecule (EpCAM) expression of exosomes were measured by NTA. Quantum dots were conjugated with anti-EpCAM to label exosomes, and the labeled exosomes were detected by NTA in fluorescent mode. The normal-cell-derived exosomes were significantly larger than those derived from malignant cells, and exosomes were successfully labeled using anti-EpCAM-conjugated quantum dots. Exosomes from different cell lines may vary in size, and exosomes might be considered as potential diagnosis biomarkers. NTA can be considered a useful, efficient, and objective method for the study of different exosomes and their unique properties in ovarian cancer.

Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification of chromosome 20.

PubMed

Jin, Yuesheng; Zhang, Hao; Tsao, Sai Wah; Jin, Charlotte; Lv, Mei; Strömbeck, Bodil; Wiegant, Joop; Wan, Thomas Shek Kong; Yuen, Po Wing; Kwong, Yok-Lam

2004-01-01

This study aimed at identifying the genetic events involved in immortalization of ovarian epithelial cells, which might be important steps in ovarian carcinogenesis. The genetic profiles of five human ovarian surface epithelial (HOSE) cell lines immortalized by retroviral transfection of the human papillomavirus (HPV) E6/E7 genes were thoroughly characterized by chromosome banding and fluorescence in situ hybridization (FISH), at various passages pre- and post-crisis. In pre-crisis, most cells had simple, non-clonal karyotypic changes. Telomere association was the commonest aberration, suggesting that tolermase dysfunction might be an important genetic event leading to cellular crisis. After immortalization post-crisis, however, the karyotypic patterns were non-random. Loss of genetic materials was a characteristic feature. The commonest numerical aberrations were -13, -14, -16, -17, -18, and +5. Among them, loss of chromosome 13 was common change observed in all lines. The only recurrent structural aberration was homogeneously staining regions (hsr) observed in three lines. FISH and combined binary ratio labeling (COBRA)-FISH showed in two cases that the hsrs were derived from chromosome 20. Clonal evolution was observed in four of the lines. In one line, hsr was the only change shared by all subclones, suggesting that it might be a primary event in cell immortalization. The results of the present study suggested that loss of chromosome 13 and the amplification of chromosome 20 might be early genetic events involved in ovarian cell immortalization, and might be useful targets for the study of genomic aberrations in ovarian carcinogenesis.

Telomerase Activity Impacts on Epstein-Barr Virus Infection of AGS Cells

PubMed Central

Rac, Jürgen; Haas, Florian; Schumacher, Andrina; Middeldorp, Jaap M.; Delecluse, Henri-Jacques; Speck, Roberto F.

2015-01-01

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva. PMID:25856387

Injurious effects of wool and grain dusts on alveolar epithelial cells and macrophages in vitro.

PubMed Central

Brown, D M; Donaldson, K

1991-01-01

Epidemiological studies of workers in wool textile mills have shown a direct relation between the concentration of wool dust in the air and respiratory symptoms. Injurious effects of wool dust on the bronchial epithelium could be important in causing inflammation and irritation. A pulmonary epithelial cell line in vitro was therefore used to study the toxic effects of wool dust. Cells of the A549 epithelial cell line were labelled with 51Cr and treated with whole wool dusts and extracts of wool, after which injury was assessed. Also, the effects of grain dust, which also causes a form of airway obstruction, were studied. The epithelial injury was assessed by measuring 51Cr release from cells as an indication of lysis, and by monitoring cells which had detached from the substratum. No significant injury to A549 cells was caused by culture with any of the dusts collected from the air but surface "ledge" dust caused significant lysis at some doses. Quartz, used as a toxic control dust, caused significant lysis at the highest concentration of 100 micrograms/well. To determine whether any injurious material was soluble the dusts were incubated in saline and extracts collected. No extracts caused significant injury to epithelial cells. A similar lack of toxicity was found when 51Cr labelled control alveolar macrophages were targets for injury. Significant release of radiolabel was evident when macrophages were exposed to quartz at concentrations of 10 and 20 micrograms/well, there being no significant injury with either wool or grain dusts. These data suggest that neither wool nor grain dust produce direct injury to epithelial cells, and further studies are necessary to explain inflammation leading to respiratory symptoms in wool and grain workers. PMID:2015211

Mullerian papilloma-like proliferation arising in cystic pelvic endosalpingiosis.

PubMed

McCluggage, W Glenn; O'Rourke, Declan; McElhenney, Clodagh; Crooks, Michael

2002-09-01

This report describes an unusual epithelial proliferation occurring in pelvic cystic endosalpingiosis. A cyst mass lined by a layer of ciliated epithelial cells involved the posterior surface of the cervix and vagina. The epithelial proliferation within the wall resembled a mullerian papilloma with fibrous and fibrovascular cores lined by bland cuboidal epithelial cells. Other areas had a microglandular growth pattern resembling cervical microglandular hyperplasia, and focally there was a solid growth pattern. Foci of typical endosalpingiosis involved the surface of both ovaries and pelvic soft tissues. The cystic lesion recurred after partial cystectomy and drainage and was followed up radiologically and with periodic fine-needle aspiration. Part of the wall of the cyst removed 11 years after the original surgery showed an identical epithelial proliferation. MIB1 staining showed a proliferation index of less than 5%, contrasting with the higher proliferation index of a typical serous borderline tumor. The differential diagnosis is discussed. As far as we are aware, this is the first report of such a benign epithelial proliferation involving cystic endosalpingiosis. Copyright 2002, Elsevier Science (USA). All rights reserved.

Toll-Like Receptor 4 Expression in the Epithelium of Inflammatory Periapical Lesions.

PubMed Central

Leonardi, R.; Perrotta, R.E.; Musumeci, G.; Crimi, S.; dos Santos, J.N.; Rusu, M.C.; Bufo, P.; Barbato, E.; Pannone, G.

2015-01-01

Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment. Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease. PMID:26708181

Toll-like receptor 4 expression in the epithelium of inflammatory periapical lesions. An immunohistochemical study.

PubMed

Leonardi, R; Perrotta, R E; Loreto, C; Musumeci, G; Crimi, S; Dos Santos, J N; Rusu, M C; Bufo, P; Barbato, E; Pannone, G

2015-10-26

Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment. Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease.

Immunohistochemical evaluation of inducible nitric oxide synthase in the epithelial lining of odontogenic cysts: A qualitative and quantitative analysis

PubMed Central

Akshatha, B K; Karuppiah, Karpagaselvi; Manjunath, G S; Kumarswamy, Jayalakshmi; Papaiah, Lokesh; Rao, Jyothi

2017-01-01

Introduction: The three common odontogenic cysts include radicular cysts (RCs), dentigerous cysts (DCs), and odontogenic keratocysts (OKCs). Among these 3 cysts, OKC is recently been classified as benign keratocystic odontogenic tumor attributing to its aggressive behavior, recurrence rate, and malignant potential. The present study involved qualitative and quantitative analysis of inducible nitric oxide synthase (iNOS) expression in epithelial lining of RCs, DCs, and OKCs, compare iNOS expression in epithelial linings of all the 3 cysts and determined overexpression of iNOS in OKCs which might contribute to its aggressive behavior and malignant potential. Aims: The present study is to investigate the role of iNOS in the pathogenesis of OKCs, DCs, and RCs by evaluating the iNOS expression in the epithelial lining of these cysts. Subjects and Methods: Analysis of iNOS expression in epithelial lining cells of 20 RCs, 20 DCs, and 20 OKCs using immunohistochemistry done. Statistical Analysis Used: The percentage of positive cells and intensity of stain was assessed and compared among all the 3 cysts using contingency coefficient. Kappa statistics for the two observers were computed for finding interobserver agreement. Results: The percentage of iNOS-positive cells was found to be remarkably high in OKCs (12/20) –57.1% as compared to RCs (6/20) – 28.6% and DCs (3/20) – 14.3%. The interobserver agreement for iNOS-positive percentage cells was arrived with kappa values with OKCs → Statistically significant (P > 0.000), RCs → statistically significant (P > 0.001) with no significant values for DCs. No statistical difference exists among 3 study samples in regard to the intensity of staining with iNOS. Conclusions: Increased iNOS expression in OKCs may contribute to bone resorption and accumulation of wild-type p53, hence, making OKCs more aggressive. PMID:29391711

The periesophageal celom of the articulate brachiopod Hemithyris psittacea (Rhynchonelliformea, Brachiopoda).

PubMed

Kuzmina, Tatyana V; Malakhov, Vladimir V

2011-02-01

The celomic system of the articulate brachiopod Hemithyris psittacea is composed of the perivisceral cavity, the canal system of the lophophore, and the periesophageal celom. We study the microscopic anatomy and ultrastructure of the periesophageal celom using scanning and transmission electron microscopy. The periesophageal celom surrounds the esophagus, is isolated from the perivisceral cavity, and is divided by septa. The lining of the periesophageal celom includes two types of cells, epithelial cells and myoepithelial cells, both are monociliary. Some epithelial cells have long processes extending along the basal lamina, suggesting that these cells might function as podocytes. The myoepithelial cells have basal myofilaments and may be overlapped by the apical processes of the adjacent epithelial cells. The periesophageal celom forms protrusions that penetrate the extracellular matrix (ECM) of the body wall above the mouth and the ECM that surrounds the esophagus. The canals of the esophageal ECM form a complicated system. The celomic lining of the external circumferential canals consists of the epithelial cells and the podocyte-like cells. The deepest canals lack a lumen; they are filled with the muscle cells surrounded by basal lamina. These branched canals might perform dual functions. First, they increase the surface area and might therefore facilitate ultrafiltration through the podocyte-like cells. Second, the deepest canals form the thickened muscle wall of the esophagus and could be necessary for antiperistalsis of the gut. Copyright © 2010 Wiley-Liss, Inc.

Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

PubMed

Berndt, Benjamin; Haverkampf, Sonja; Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

PubMed Central

Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S.; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. PMID:23667660

TLR3-mediated NF-{kappa}B signaling in human esophageal epithelial cells.

PubMed

Lim, Diana M; Narasimhan, Sneha; Michaylira, Carmen Z; Wang, Mei-Lun

2009-12-01

Despite its position at the front line against ingested pathogens, very little is presently known about the role of the esophageal epithelium in host innate immune defense. As a key player in the innate immune response, Toll-like receptor (TLR) signaling has not been well characterized in human esophageal epithelial cells. In the present study, we investigated the inflammatory response and signaling pathways activated by TLR stimulation of human esophageal cells in vitro. Using quantitative RT-PCR, we profiled the expression pattern of human TLRs 1-10 in primary esophageal keratinocytes (EPC2), immortalized nontransformed esophageal keratinocytes (EPC2-hTERT), and normal human esophageal mucosal biopsies and found that TLRs 1, 2, 3, and 5 were expressed both in vivo and in vitro. Using the cytokine IL-8 as a physiological read out of the inflammatory response, we found that TLR3 is the most functional of the expressed TLRs in both primary and immortalized esophageal epithelial cell lines in response to its synthetic ligand polyinosinic polycytidylic acid [poly(I:C)]. Through reporter gene studies, we show that poly(I:C)-induced NF-kappaB activation is critical for the transactivation of the IL-8 promoter in vitro and that nuclear translocation of NF-kappaB occurs at an early time point following poly(I:C) stimulation of esophageal epithelial cells. Importantly, we also show that poly(I:C) stimulation induces the NF-kappaB-dependent esophageal epithelial expression of TLR2, leading to enhanced epithelial responsiveness of EPC2-hTERT cells to TLR2 ligand stimulation, suggesting an important regulatory role for TLR3-mediated NF-kappaB signaling in the innate immune response of esophageal epithelial cells. Our findings demonstrate for the first time that TLR3 is highly functional in the human esophageal epithelium and that TLR3-mediated NF-kappaB signaling may play an important regulatory role in esophageal epithelial homeostasis.

The influence of matrix properties on growth and morphogenesis of human pancreatic ductal epithelial cells in 3D

PubMed Central

Raza, Asad; Ki, Chang Seok; Lin, Chien-Chi

2013-01-01

A highly tunable synthetic biomimetic hydrogel platform was developed to study the growth and morphogenesis of pancreatic ductal epithelial cells (PDEC) under the influence of a myriad of instructive cues. A PDEC line, PANC-1, was used as a model system to illustrate the importance of matrix compositions on cell fate determination. PANC-1 is an immortalized ductal epithelial cell line widely used in the study of pancreatic tumor cell behaviors. PANC-1 cells are also increasingly explored as a potential cell source for endocrine differentiation. Thus far, most studies related to PANC-1, among other PDEC lines, are performed on 2D culture surfaces. Here, we evaluated the effect of matrix compositions on PANC-1 cell growth and morphogenesis in 3D. Specifically, PANC-1 cells were encapsulated in PEG-based hydrogels prepared by step-growth thiol-ene photopolymerization. It was found that thiol-ene hydrogels provided a cytocompatible environment for encapsulation and 3D culture of PANC-1 cells. In contrast to a monolayer morphology on 2D culture surfaces, PANC-1 cells formed clusters in 3D thiol-ene hydrogels within 4 days of culture. After culturing for 10 days, however, the growth and structures of these clusters were significantly impacted by gel matrix properties, including sensitivity of the matrix to proteases, stiffness of the matrix, and ECM-mimetic motifs. The use of matrix metalloproteinase (MMP) sensitive linker or the immobilization of fibronectin-derived RGDS ligand in the matrix promoted PANC-1 cell growth and encouraged them to adopt ductal cyst-like structures. On the other hand, the encapsulated cells formed smaller and more compact aggregates in non-MMP responsive gels. The incorporation of laminin-derived YIGSR peptide did not enhance cell growth and caused the cells to form compact aggregates. Immobilized YIGSR also enhanced the expression of epithelial cell markers including β-catenin and E-cadherin. These studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for studying and manipulating pancreatic epithelial cell growth and morphogenesis in 3D. PMID:23602364

Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.

PubMed

Stamps, A C; Davies, S C; Burman, J; O'Hare, M J

1994-06-15

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells

PubMed Central

Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N.; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C.

2013-01-01

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling. PMID:23700468

Gene-expression profiles of epithelial cells treated with EMD in vitro: analysis using complementary DNA arrays.

PubMed

Kapferer, I; Schmidt, S; Gstir, R; Durstberger, G; Huber, L A; Vietor, I

2011-02-01

During surgical periodontal treatment, EMD is topically applied in order to facilitate regeneration of the periodontal ligament, acellular cementum and alveolar bone. Suppresion of epithelial down-growth is essential for successful periodontal regeneration; however, the underlying mechanisms of how EMD influences epithelial wound healing are poorly understood. In the present study, the effects of EMD on gene-expression profiling in an epithelial cell line (HSC-2) model were investigated. Gene-expression modifications, determined using a comparative genome-wide expression-profiling strategy, were independently validated by quantitative real-time RT-PCR. Additionally, cell cycle, cell growth and in vitro wound-healing assays were conducted. A set of 43 EMD-regulated genes was defined, which may be responsible for the reduced epithelial down-growth upon EMD application. Gene ontology analysis revealed genes that could be attributed to pathways of locomotion, developmental processes and associated processes such as regulation of cell size and cell growth. Additionally, eight regulated genes have previously been reported to take part in the process of epithelial-to-mesenchymal transition. Several independent experimental assays revealed significant inhibition of cell migration, growth and cell cycle by EMD. The set of EMD-regulated genes identified in this study offers the opportunity to clarify mechanisms underlying the effects of EMD on epithelial cells. Reduced epithelial repopulation of the dental root upon periodontal surgery may be the consequence of reduced migration and cell growth, as well as epithelial-to-mesenchymal transition. © 2010 John Wiley & Sons A/S.

Complex Determinants of Epithelial: Mesenchymal Phenotypic Plasticity in Ovarian Cancer

PubMed Central

Klymenko, Yuliya; Kim, Oleg; Stack, M. Sharon

2017-01-01

Unlike most epithelial malignancies which metastasize hematogenously, metastasis of epithelial ovarian cancer (EOC) occurs primarily via transcoelomic dissemination, characterized by exfoliation of cells from the primary tumor, avoidance of detachment-induced cell death (anoikis), movement throughout the peritoneal cavity as individual cells and multi-cellular aggregates (MCAs), adhesion to and disruption of the mesothelial lining of the peritoneum, and submesothelial matrix anchoring and proliferation to generate widely disseminated metastases. This exceptional microenvironment is highly permissive for phenotypic plasticity, enabling mesenchymal-to-epithelial (MET) and epithelial-to-mesenchymal (EMT) transitions. In this review, we summarize current knowledge on EOC heterogeneity in an EMT context, outline major regulators of EMT in ovarian cancer, address controversies in EMT and EOC chemoresistance, and highlight computational modeling approaches toward understanding EMT/MET in EOC. PMID:28792442

Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture.

PubMed

Deora, Ami A; Diaz, Fernando; Schreiner, Ryan; Rodriguez-Boulan, Enrique

2007-10-01

Electroporation-mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well-established procedures to electroporate polarized epithelial cells adhering to a tissue culture substrate (glass, plastic or filter). We report here the development of a simple procedure that uses available commercial equipment and works efficiently and reproducibly for a variety of epithelial cell lines in culture.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

PubMed

Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential.

PubMed

Green, K J; Stappenbeck, T S; Noguchi, S; Oyasu, R; Nilles, L A

1991-03-01

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.

Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878).

PubMed

Soni, Pankaj; Pradhan, Pravata K; Swaminathan, T R; Sood, Neeraj

2018-06-01

A cell line, designated as PHF, has been established from caudal fin of Pangasianodon hypophthalmus. The cell line was developed using explant method and PHF cells have been subcultured for more than 72 passages over a period of 14 months. The cells were able to grow at temperatures between 24 and 32° C, with an optimum temperature of 28° C. The growth rate of PHF cells was directly proportional to FBS concentration, with optimum growth observed at 20% FBS concentration. On the basis of immunophenotyping assay, PHF cells were confirmed to be of epithelial type. Karyotyping of PHF cells revealed diploid number of chromosomes (2n = 60) at 39th and 65th passage, which indicated that the developed cell line is chromosomally stable. The origin of the cell line was confirmed by amplification and sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. The cell line was tested for Mycoplasma contamination and found to be negative. The cells were successfully transfected with GFP reporter gene suggesting that the developed cell line could be utilized for gene expression studies in future. The cell line could be successfully employed for evaluating the cytotoxicity of heavy metals, namely mercuric chloride and sodium arsenite suggesting that PHF cell line can be potential surrogate for whole fish for studying the cytotoxicity of water soluble compounds. The result of virus susceptibility to tilapia lake virus (TiLV) revealed that PHF cells were refractory to TiLV virus. The newly established cell line would be a useful tool for investigating disease outbreaks particularly of viral etiology, transgenic as well as cytotoxicity studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Cytokine Expression and Production by Purified Helicobacter pylori Urease in Human Gastric Epithelial Cells

PubMed Central

Tanahashi, Toshihito; Kita, Masakazu; Kodama, Tadashi; Yamaoka, Yoshio; Sawai, Naoki; Ohno, Tomoyuki; Mitsufuji, Shoji; Wei, Ya-Ping; Kashima, Kei; Imanishi, Jiro

2000-01-01

Cytokines have been proposed to play an important role in Helicobacter pylori-associated gastroduodenal diseases, but the exact mechanism of the cytokine induction remains unclear. H. pylori urease, a major component of the soluble proteins extracted from bacterial cells, is considered to be one of the virulence factors for the inflammation in the gastric mucosa that is produced in H. pylori infection. However, the response of human gastric epithelial cells to the stimulation of urease has not been investigated. In the present study, we used human gastric epithelial cells in a primary culture system and examined whether H. pylori urease stimulates the gastric epithelial cells to induce proinflammatory cytokines by reverse transcription-PCR and enzyme-linked immunosorbent assay. First, by using peripheral blood mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells), we confirmed the ability of purified H. pylori urease to induce the production of proinflammatory cytokines. Furthermore, we demonstrated that the human gastric epithelial cells produced interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8, following stimulation with purified urease. The patterns of cytokine induction differed among human PBMC, MKN-45 cells, and human gastric epithelial cells. These results suggest that the human gastric epithelial cells contribute to the induction of proinflammatory cytokines by the stimulation of H. pylori urease, indicating that the epithelial cells were involved in the mucosal inflammation that accompanied H. pylori infection. PMID:10639431

An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

PubMed

Shiao, Yih-Horng; Lupascu, Sorin T; Gu, Yuhan D; Kasprzak, Wojciech; Hwang, Christopher J; Fields, Janet R; Leighty, Robert M; Quiñones, Octavio; Shapiro, Bruce A; Alvord, W Gregory; Anderson, Lucy M

2009-10-19

Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease.

PubMed

Mori, Nozomu; Miyashita, Takenori; Inamoto, Ryuhei; Matsubara, Ai; Mori, Terushige; Akiyama, Kosuke; Hoshikawa, Hiroshi

2017-04-01

Ion transport and its regulation in the endolymphatic sac (ES) are reviewed on the basis of recent lines of evidence. The morphological and physiological findings demonstrate that epithelial cells in the intermediate portion of the ES are more functional in ion transport than those in the other portions. Several ion channels, ion transporters, ion exchangers, and so on have been reported to be present in epithelial cells of ES intermediate portion. An imaging study has shown that mitochondria-rich cells in the ES intermediate portion have a higher activity of Na + , K + -ATPase and a higher Na + permeability than other type of cells, implying that molecules related to Na + transport, such as epithelial sodium channel (ENaC), Na + -K + -2Cl - cotransporter 2 (NKCC2) and thiazide-sensitive Na + -Cl - cotransporter (NCC), may be present in mitochondria-rich cells. Accumulated lines of evidence suggests that Na + transport is most important in the ES, and that mitochondria-rich cells play crucial roles in Na + transport in the ES. Several lines of evidence support the hypothesis that aldosterone may regulate Na + transport in ES, resulting in endolymph volume regulation. The presence of molecules related to acid/base transport, such as H + -ATPase, Na + -H + exchanger (NHE), pendrin (SLC26A4), Cl - -HCO 3 - exchanger (SLC4A2), and carbonic anhydrase in ES epithelial cells, suggests that acid/base transport is another important one in the ES. Recent basic and clinical studies suggest that aldosterone may be involved in the effect of salt-reduced diet treatment in Meniere's disease.

Evidence from a Mouse Model That Epithelial Cell Migration and Mesenchymal-Epithelial Transition Contribute to Rapid Restoration of Uterine Tissue Integrity during Menstruation

PubMed Central

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

2014-01-01

Background In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. Methodology A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Principal Findings Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. Conclusions/Significance These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These insights may inform development of new therapies to induce rapid healing in the endometrium and other tissues and offer hope to women who suffer from heavy menstrual bleeding. PMID:24466063

Depleted uranium induces neoplastic transformation in human lung epithelial cells.

PubMed

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

2010-02-15

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

Solitomab, an epithelial cell adhesion molecule/CD3 bispecific antibody (BiTE), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo.

PubMed

English, Diana P; Bellone, Stefania; Schwab, Carlton L; Roque, Dana M; Lopez, Salvatore; Bortolomai, Ileana; Cocco, Emiliano; Bonazzoli, Elena; Chatterjee, Sudeshna; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Rutherford, Thomas J; Santin, Alessandro D

2015-02-01

Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM. © 2014 American Cancer Society.

In Vitro Study of Influence of Au Nanoparticles on HT29 and SPEV Cell Lines

NASA Astrophysics Data System (ADS)

Pavlovich, Elena; Volkova, Nataliia; Yakymchuk, Elena; Perepelitsyna, Olena; Sydorenko, Michail; Goltsev, Anatoliy

2017-08-01

Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6-12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1-3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6-12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment.

Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation*

PubMed Central

Uchida, Hiroshi; Maruyama, Tetsuo; Nishikawa-Uchida, Sayaka; Oda, Hideyuki; Miyazaki, Kaoru; Yamasaki, Akiko; Yoshimura, Yasunori

2012-01-01

Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin. PMID:22174415

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

PubMed

Chiba, Eriko; Villena, Julio; Hosoya, Shoichi; Takanashi, Naoya; Shimazu, Tomoyuki; Aso, Hisashi; Tohno, Masanori; Suda, Yoshihito; Kawai, Yasushi; Saito, Tadao; Miyazawa, Kenji; He, Fang; Kitazawa, Haruki

2012-10-01

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts. Copyright © 2011 Elsevier Ltd. All rights reserved.

Generation of Mouse Lung Epithelial Cells.

PubMed

Kasinski, Andrea L; Slack, Frank J

2013-08-05

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 Kras LSL-G12D/+ ; p53 LSL-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 p53 R172 . 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.

CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS

PubMed Central

Dang, Xitong; Eliceiri, Brian P.; Baird, Andrew; Costantini, Todd W.

2015-01-01

The human genome contains a unique, distinct, and human-specific α7-nicotinic acetylcholine receptor (α7nAChR) gene [CHRNA7 (gene-encoding α7-nicotinic acetylcholine receptor)] called CHRFAM7A (gene-encoding dup-α7-nicotinic acetylcholine receptor) on a locus of chromosome 15 associated with mental illness, including schizophrenia. Located 5′ upstream from the “wild-type” CHRNA7 gene that is found in other vertebrates, we demonstrate CHRFAM7A expression in a broad range of epithelial cells and sequenced the CHRFAM7A transcript found in normal human fetal small intestine epithelial (FHs) cells to prove its identity. We then compared its expression to CHRNA7 in 11 gut epithelial cell lines, showed that there is a differential response to LPS when compared to CHRNA7, and characterized the CHRFAM7A promoter. We report that both CHRFAM7A and CHRNA7 gene expression are widely distributed in human epithelial cell lines but that the levels of CHRFAM7A gene expression vary up to 5000-fold between different gut epithelial cells. A 3-hour treatment of epithelial cells with 100 ng/ml LPS increased CHRFAM7A gene expression by almost 1000-fold but had little effect on CHRNA7 gene expression. Mapping the regulatory elements responsible for CHRFAM7A gene expression identifies a 1 kb sequence in the UTR of the CHRFAM7A gene that is modulated by LPS. Taken together, these data establish the presence, identity, and differential regulation of the human-specific CHRFAM7A gene in human gut epithelial cells. In light of the fact that CHRFAM7A expression is reported to modulate ligand binding to, and alter the activity of, the wild-type α7nAChR ligand-gated pentameric ion channel, the findings point to the existence of a species-specific α7nAChR response that might regulate gut epithelial function in a human-specific fashion.—Dang, X., Eliceiri, B. P., Baird, A., Costantini, T. W. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS. PMID:25681457

Development of human epithelial cell systems for radiation risk assessment

NASA Astrophysics Data System (ADS)

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

1994-10-01

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

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.

Airway epithelial cell response to human metapneumovirus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, X.; Liu, T.; Spetch, L.

2007-11-10

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

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

PubMed Central

Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

2002-01-01

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID:11772392

Progesterone-induced miR-133a inhibits the proliferation of endometrial epithelial cells.

PubMed

Pan, J-L; Yuan, D-Z; Zhao, Y-B; Nie, L; Lei, Y; Liu, M; Long, Y; Zhang, J-H; Blok, L J; Burger, C W; Yue, L-M

2017-03-01

This study aimed to understand the role of miR-133a in progesterone actions, explore the regulative mechanism of the progesterone receptor, and investigate the effects of miR-133a on the progesterone-inhibited proliferation of mouse endometrial epithelial cells. The expression of miR-133a induced by progesterone was detected by quantitative real-time PCR both in vivo and in vitro. Ishikawa subcell lines stably transfected with progesterone receptor subtypes were used to determine the receptor mechanism of progesterone inducing miR-133a. Specific miR-133a mimics or inhibitors were transfected into mouse uteri and primary cultured endometrial epithelial cells to overexpress or downregulate the miR-133a. The roles of miR-133a in the cell cycle and proliferation of endometrial epithelial cells were analysed by flow cytometry and Edu incorporation analysis. The protein levels of cyclinD2 in uterine tissue sections and primary cultured endometrial epithelial cells were determined by immunohistochemistry and Western blot analysis. Progesterone could induce miR-133a expression in a PRB-dependent manner in endometrial epithelial cells. miR-133a inhibited endometrial epithelial cell proliferation by arresting cell cycle at the G 1 -S transition. Moreover, miR-133a acted as an inhibitor in downregulating cyclinD2 in endometrial epithelial cells. We showed for the first time that progesterone-induced miR-133a inhibited the proliferation of endometrial epithelial cells by downregulating cyclinD2. Our research indicated an important mechanism for progesterone inhibiting the proliferation of endometrial epithelial cells by inducing special miRNAs to inhibit positive regulatory proteins in the cell cycle. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Establishment and characterization of a new human acinar cell carcinoma cell line, Faraz-ICR, from pancreas.

PubMed

Rezaei, Marzieh; Hosseini, Ahmad; Nikeghbalian, Saman; Ghaderi, Abbas

Basic research in the field of acinar cell carcinoma (ACC) as a rare neoplasm of the pancreas is dependent on the availability of pragmatic model such as new pancreatic cancer cell lines. Thus, establishment and characterization of new pancreatic cancer cell lines from ACC origin are deemed important. Faraz-ICR cell line was derived from a 58-years old woman with pancreatic acinar cell carcinoma by the collagenase digestion protocol. We characterized the cell line by examining its morphology and cytostructural and functional profile. Faraz-ICR has a doubling time of 35 hours and grows in soft agar with a colony-forming efficiency of 25%. The cell had nearly normal pattern of chromosomes in karyotype analysis and Comparative Genomic Hybridization (CGH) array analysis. Evaluation of cells by flowcytometry showed that Faraz-ICR is negative for EpCAM and mesenchymal markers in different passages, and has epithelial nature. Immunofluorescence staining revealed that cells were strongly positive for vimentin, desmin, ezrin, S100, nestin and they were negative for pan-cytokeratins, chromogranin and alpha smooth muscle actin. We were able to establish a new pancreatic carcinoma cell line with partial aspects of Epithelial-mesenchymal transition and aggressiveness. This cell line might be suitable for studying various anticancer drugs and protein profile aiming to see any possible tumor associated marker for ACC. Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Establishment and characterization of Asian oral cancer cell lines as in vitro models to study a disease prevalent in Asia.

PubMed

Hamid, Sharifah; Lim, Kue Peng; Zain, Rosnah Binti; Ismail, Siti Mazlipah; Lau, Shin Hin; Mustafa, Wan Mahadzir Wan; Abraham, M Thomas; Nam, Noor Akmar; Teo, Soo-Hwang; Cheong, Sok Ching

2007-03-01

We have established 3 cell lines ORL-48, -115 and -136 from surgically resected specimens obtained from untreated primary human oral squamous cell carcinomas of the oral cavity. The in vitro growth characteristics, epithelial origin, in vitro anchorage independency, human papilloma-virus (HPV) infection, microsatellite instability status, karyotype and the status of various cell cycle regulators and gatekeepers of these cell lines were investigated. All 3 cell lines grew as monolayers with doubling times ranging between 26.4 and 40.8 h and were immortal. Karyotyping confirmed that these cell lines were of human origin with multiple random losses and gains of entire chromosomes and regions of chromosomes. Immunohistochemistry staining of cytokeratins confirmed the epithelial origin of these cell lines, and the low degree of anchorage independency expressed by these cell lines suggests non-transformed phenotypes. Genetic analysis identified mutations in the p53 gene in all cell lines and hypermethylation of p16INK4a in ORL-48 and -136. Analysis of MDM2 and EGFR expression indicated MDM2 overexpression in ORL-48 and EGFR overexpression in ORL-136 in comparison to the protein levels in normal oral keratinocytes. Analysis of the BAT-26 polyadenine repeat sequence and MLH-1 and MSH-2 repair enzymes demonstrated that all 3 cell lines were microsatellite stable. The role of HPV in driving carcinogenesis in these tumours was negated by the absence of HPV. Finally, analysis of the tissues from which these cell lines were derived indicated that the cell lines were genetically representative of the tumours, and, therefore, are useful tools in the understanding of the molecular changes associated with oral cancers.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harkema, J.R.; Hotchkiss, J.A.; Griffith, W.C.

The present study was designed to examine the effects of long-term ozone exposure on nasal epithelia and intraepithelial mucosubstances (IM) throughout the nasal airways of F344/N rats. Animals were exposed to 0 (controls). 0. 12. 0.5, or 1.0 ppm ozone. 6 h/day, 5 days/wk. for 20 mo. Rats were killed 1 wk after the end of the exposure. and nasal tissues were processed for light and electron microscopy. Standard morphometric techniques were used to determine epithelial cell densities and the amounts of IM in the surface epithelium lining the nasal airways. No mucous cells or IM were present in themore » epithelia lining the nasal lateral meatus and maxillary sinus of rats exposed to 0 or 0.12 ppm ozone. In contrast, rats exposed to 0.5 or 1.0 ppm ozone had marked mucous cell metaplasia (MCM) with numerous mucous cells and conspicuous amounts of IM in the surface epithelium lining these upper airways. Ozone-induced increases in total epithelial cells (i.e., epithelial hyperplasia) were present only in rats exposed to 1.0 ppm. The results of this study indicate that rats chronically exposed to 1.0 or 0.5 ppm, but not 0. 121 ppm. ozone can develop marked MCM with significant increases in IM in both proximal and distal nasal airways. The epithelial chances observed throughout the nasal passages of ozone-exposed rats may be adaptive responses in an attempt to protect the upper and lower respiratory tract from further ozone-induced injury.« less

Menadione induces the formation of reactive oxygen species and depletion of GSH-mediated apoptosis and inhibits the FAK-mediated cell invasion.

PubMed

Kim, Yun Jeong; Shin, Yong Kyoo; Sohn, Dong Suep; Lee, Chung Soo

2014-09-01

Menadione induces apoptosis in tumor cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to menadione is not clear. In addition, it is unclear whether menadione-induced apoptosis is mediated by the depletion of glutathione (GSH) contents that is associated with the formation of reactive oxygen species. Furthermore, the effect of menadione on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of menadione exposure on apoptosis, cell adhesion, and cell migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that menadione may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of menadione appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Menadione inhibited fetal-bovine-serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase (FAK)-dependent activation of cytoskeletal-associated components. Therefore, menadione might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy.

Breakdown evaluation of corneal epithelial barrier caused by antiallergic eyedrops using an electrophysiologic method.

PubMed

Nakashima, Mikiro; Nakamura, Tadahiro; Teshima, Mugen; To, Hideto; Uematsu, Masafumi; Kitaoka, Takashi; Taniyama, Kotaro; Nishida, Koyo; Nakamura, Junzo; Sasaki, Hitoshi

2008-02-01

The aim of this study was to examine the usefulness of an electrophysiologic method for predicting corneal epithelial breakdown by antiallergic eyedrops and comparing the results with those in other appraisal methods. Six kinds of antiallergic eyedrops, including benzalkonium chloride (BK) as an ophthalmic preservative and two kinds of BK-free antiallergic eyedrops, were used in this study. Eyedrops were applied to excise rabbit corneas and monitoring was performed according to an electrophysiologic method, using a commercially available chamber system to mimic human tear turnover. Changes in transepithelial electrical resistance (TEER) in the corneal surface were recorded. The cytotoxicity of each kind of eyedrops in a normal rabbit corneal epithelial (NRCE) cell line and a human endothelial cell line EA.hy926 was also examined. The extent of decrease in the corneal TEER after applying antiallergic eyedrops was dependent on the concentration of the BK included as a preservative, but it was also affected by the different kinds of drugs when the BK concentration was low. Higher cytotoxicity of the eyedrops against the NRCE and EA.hy926 cell lines was observed with a reduction of TEER. Monitoring changes in the corneal TEER, according to the electrophysiologic method with the application of antiallergic eyedrops, is useful for predicting corneal epithelial breakdown caused by their instillation.

Pseudomonas aeruginosa LasB protease impairs innate immunity in mice and humans by targeting a lung epithelial cystic fibrosis transmembrane regulator–IL-6–antimicrobial–repair pathway

PubMed Central

Saint-Criq, Vinciane; Villeret, Bérengère; Bastaert, Fabien; Kheir, Saadé; Hatton, Aurélie; Cazes, Aurélie; Xing, Zhou; Sermet-Gaudelus, Isabelle; Garcia-Verdugo, Ignacio; Edelman, Aleksander

2018-01-01

Background Pseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity. Objective We evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo. Methods Wild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models. Results We showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways. Conclusions Our data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals. PMID:28790180

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. © 2010 Blackwell Verlag GmbH.

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

PubMed Central

Huang, G T; Eckmann, L; Savidge, T C; Kagnoff, M F

1996-01-01

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens. PMID:8755670

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells.

PubMed

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

2005-11-18

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

A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

PubMed

Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

2014-12-01

Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

Stomatin-like protein 2 is overexpressed in epithelial ovarian cancer and predicts poor patient survival.

PubMed

Sun, Fei; Ding, Wen; He, Jie-Hua; Wang, Xiao-Jing; Ma, Ze-Biao; Li, Yan-Fang

2015-10-20

Stomatin-like protein 2 (SLP-2, also known as STOML2) is a stomatin homologue of uncertain function. SLP-2 overexpression has been suggested to be associated with cancer progression, resulting in adverse clinical outcomes in patients. Our study aim to investigate SLP-2 expression in epithelial ovarian cancer cells and its correlation with patient survival. SLP-2 mRNA and protein expression levels were analysed in five epithelial ovarian cancer cell lines and normal ovarian epithelial cells using real-time PCR and western blotting analysis. SLP-2 expression was investigated in eight matched-pair samples of epithelial ovarian cancer and adjacent noncancerous tissues from the same patients. Using immunohistochemistry, we examined the protein expression of paraffin-embedded specimens from 140 patients with epithelial ovarian cancer, 20 cases with borderline ovarian tumours, 20 cases with benign ovarian tumours, and 20 cases with normal ovarian tissues. Statistical analyses were applied to evaluate the clinicopathological significance of SLP-2 expression. SLP-2 mRNA and protein expression levels were significantly up-regulated in epithelial ovarian cancer cell lines and cancer tissues compared with normal ovarian epithelial cells and adjacent noncancerous ovarian tissues. Immunohistochemistry analysis revealed that the relative overexpression of SLP-2 was detected in 73.6 % (103/140) of the epithelial ovarian cancer specimens, 45.0 % (9/20) of the borderline ovarian specimens, 30.0 % (6/20) of the benign ovarian specimens and none of the normal ovarian specimens. SLP-2 protein expression in epithelial ovarian cancer was significantly correlated with the tumour stage (P < 0.001). Epithelial ovarian cancer patients with higher SLP-2 protein expression levels had shorter progress free survival and overall survival times compared to patients with lower SLP-2 protein expression levels. Multivariate analyses showed that SLP-2 expression levels were an independent prognostic factor for survival in epithelial ovarian cancer patients. SLP-2 mRNA and proteins were overexpressed in epithelial ovarian cancer tissues. SLP-2 protein overexpression was associated with advanced stage disease. Patients with higher SLP-2 protein expression had shorter progress free survival and poor overall survival times. Thus, SLP-2 protein expression was an independent prognostic factor for patients with epithelial ovarian cancer.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

PubMed

Easty, D J; Guthrie, B A; Maung, K; Farr, C J; Lindberg, R A; Toso, R J; Herlyn, M; Bennett, D C

1995-06-15

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

Optimization of FNAC findings as a preoperative diagnostic aid for odontogenic cysts.

PubMed

Jain, Garima; Shetty, Pushparaja

2015-01-01

Fine-needle aspiration cytology (FNAC) is not a definitive preoperative diagnostic procedure done for all cases of odontogenic cysts. This is because of the inconsistent results obtained with it. This study was done to optimize FNAC findings and help in preoperative characterization of odontogenic cysts. Cystic fluid was collected and centrifuged from 50 odontogenic cysts that were planned for excision. Three smears were prepared from the cell sediment obtained after centrifugation and stained. The stained sections were examined for presence and type of epithelial cells, to formulate a preopererative diagnosis. Epithelial cells were detected in 46% cases in smear 1, 48% cases in smear 2, and 52% cases in smear 3. When all three smears from one case were studied, 86% cases showed epithelial cells for evaluation. Cystic aspirate should be centrifuged and the entire cell sediment should be examined by making multiple smears for evaluation of cystic epithelial lining cells.

Gene Transfer by Guanidinium-Cholesterol Cationic Lipids into Airway Epithelial Cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Oudrhiri, Noufissa; Vigneron, Jean-Pierre; Peuchmaur, Michel; Leclerc, Tony; Lehn, Jean-Marie; Lehn, Pierre

1997-03-01

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis (guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

Establishment of two basal-like breast cancer cell lines with extremely low tumorigenicity from Taiwanese premenopausal women.

PubMed

Kuo, Wen-Ling; Ueng, Shir-Hwa; Wu, Chun-Hsing; Lee, Li-Yu; Lee, Yun-Shien; Yu, Ming-Chin; Chen, Shin-Cheh; Yu, Chi-Chang; Tsai, Chi-Neu

2018-04-01

The research of carcinogenetic mechanisms of breast cancer in different ethnic backgrounds is an interesting field, as clinical features of breast cancers vary among races. High premenopausal incidence is distinctive in East-Asian breast cancer. However, human cell lines derived from Asian primary breast tumor are rare. To provide alternative cell line models with a relevant genetic background, we aimed to establish breast cancer cell lines from Taiwanese patients of Han-Chinese ethnicity. Fresh tissue from mammary tumors were digested into organoids, plated and grown in basal serum-free medium of human mammary epithelial cells (HuMEC) with supplements. Cells were further enriched by positive selection with CD326 (epithelial cell adhesion molecule; EpCAM)-coated micro-magnetic beads. Two breast cancer cell lines derived from premenopausal women were successfully established by this method, and named Chang-Gung Breast Cancer 01 (CGBC 01) and 02 (CGBC 02). These two cell lines had a similar phenotype with weak expression of estrogen receptor (ER), progesterone receptor (PR), and without amplification of receptor tyrosine protein kinase erbB-2 (HER2/neu). Genome-wide Single Nucleotide Polymorphism (SNP) array showed multiple copy number alterations in both cell lines. Based on gene expression profiles, CGBC 01 and 02 were clustered into basal-like subtype with reference to the breast cancer cell line gene expression database. The tumorigenicity of both cell lines was extremely low in both anchorage-independence assay and transplantation into the mammary fat pads of nude mice. CGBC 01 and CGBC 02 are low tumorigenic breast cancer cell lines, established from Han-Chinese premenopausal breast cancer patients, which serve as in vitro models in studying the biological features of Asian breast cancer.

In vitro model for Campylobacter pylori adherence properties.

PubMed Central

Neman-Simha, V; Mégraud, F

1988-01-01

The adherence of 12 strains of Campylobacter pylori was studied on four cell lines. Immunofluorescence and scanning and transmission electron microscopy were used to visualize the bacteria. A heavy adherence to the epithelial cell line HEp-2 and to the intestinal cell line Int-407 was noted. By transmission electron microscopy, a close association between bacteria and cells in the form of cup-like structures was observed, but pedestals were not present. Images PMID:3182085

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

Human Corneal Limbal-Epithelial Cell Response to Varying Silk Film Geometric Topography In Vitro

PubMed Central

Lawrence, Brian D.; Pan, Zhi; Liu, Aihong; Kaplan, David L.; Rosenblatt, Mark I.

2012-01-01

Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal-epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment, and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36%–54% increase on average in initial cell attachment, which corresponded to an over 2-fold increase in FA localization when compared to other silk film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. In conclusion, silk film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially in regards to FA distribution, in vitro. PMID:22705042

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu Yongpeng; Li Hongzhen; Miki, Jun

2006-04-01

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferativemore » capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.« less

Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

PubMed

Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

2008-09-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Evidence for epithelial-mesenchymal transition in cancer stem-like cells derived from carcinoma cell lines of the cervix uteri.

PubMed

Lin, Jiaying; Liu, Xishi; Ding, Ding

2015-01-01

The cancer stem cell (CSC) paradigm is one possible way to understand the genesis of cancer, and cervical cancer in particular. We quantified and enriched ALDH1(+) cells within cervical cancer cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). ALDH1 expression in spheroid-derived cells (SDC) and the parental monolayer-derived cell (MDC) line was compared by flow-cytometry. Invasion capability was evaluated by Matrigel assay and expression of EMT-related genes Twist 1, Twist 2, Snail 1, Snail 2, Vimentin and E-cadherin by real-time PCR. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. SDC expressed lower levels of E-cadherin and elevated levels of Twist 1, Twist 2, Snail 1, Snail 2 and Vimentin compared to MDC. Cervical cancer cell lines harbor potential CSC, characterized by ALDH1 expression as well as properties like invasiveness, colony-forming ability, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

Epithelial-Mesenchymal Transition in Tissue Repair and Fibrosis

PubMed Central

Stone, Rivka C.; Pastar, Irena; Ojeh, Nkemcho; Chen, Vivien; Liu, Sophia; Garzon, Karen I.; Tomic-Canic, Marjana

2016-01-01

Epithelial-mesenchymal transition (EMT) describes the global process by which stationary epithelial cells undergo phenotypic changes, including loss of cell-cell adhesion and apical-basal polarity, and acquire mesenchymal characteristics which confer migratory capacity. EMT and its converse, MET (mesenchymal-to-epithelial transition), are integral stages of many physiologic processes, and as such are tightly coordinated by a host of molecular regulators. Converging lines of evidence have identified EMT as a component of cutaneous wound healing, during which otherwise stationary keratinocytes - the resident skin epithelial cells - migrate across the wound bed to restore the epidermal barrier. Moreover, EMT also plays a role in the development of scarring and fibrosis, as the matrix-producing myofibroblast arises from cells of epithelial lineage in response to injury but is pathologically sustained instead of undergoing MET or apoptosis. In this review, we summarize the role of EMT in physiologic repair and pathologic fibrosis of tissues and organs. We conclude that further investigation into the contribution of EMT to the impaired repair of fibrotic wounds may identify components of EMT signaling as common therapeutic targets for impaired healing in many tissues. PMID:27461257

Airway epithelial repair in health and disease: Orchestrator or simply a player?

PubMed

Iosifidis, Thomas; Garratt, Luke W; Coombe, Deirdre R; Knight, Darryl A; Stick, Stephen M; Kicic, Anthony

2016-04-01

Epithelial cells represent the most important surface of contact in the body and form the first line of defence of the body to external environment. Consequently, epithelia have numerous roles in order to maintain a homeostatic defence barrier. Although the epithelium has been extensively studied over several decades, it remains the focus of new research, indicating a lack of understanding that continues to exist around these cells in specific disease settings. Importantly, evidence is emerging that airway epithelial cells in particular have varied complex functions rather than simple passive roles. One area of current interest is its role following injury. In particular, the epithelial-specific cellular mechanisms regulating their migration during wound repair remain poorly understood and remain an area that requires much needed investigation. A better understanding of the physiological, cellular and molecular wound repair mechanisms could assist in elucidating pathological processes that contribute to airway epithelial pathology. This review attempts to highlight migration-specific and cell-extracellular matrix (ECM) aspects of repair used by epithelial cells under normal and disease settings, in the context of human airways. © 2016 Asian Pacific Society of Respirology.

Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor.

PubMed

Ingram, M; Techy, G B; Saroufeem, R; Yazan, O; Narayan, K S; Goodwin, T J; Spaulding, G F

1997-06-01

Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated. The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture. Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm. Architecture of spheroids varied with cell type. Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures. Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs. Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells. Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated.

Mitochondria are targets for the antituberculosis drug rifampicin in cultured epithelial cells.

PubMed

Erokhina, M V; Kurynina, A V; Onishchenko, G E

2013-10-01

Rifampicin is a widely used drug for antituberculosis therapy. Its target is the bacterial RNA polymerase. After entry into the human or mammalian organism, rifampicin is accumulated in cells of epithelial origin (kidneys, liver, lungs) where it induces apoptosis, necrosis, and fibrosis. The purpose of this study was to determine the intracellular mechanisms leading to rifampicin-induced pathological changes and cell death. We analyzed the survival and state of the chondriome of cultured epithelial cells of the SPEV line under the influence of rifampicin. Our data show that the drug induces pronounced pathological changes in the network and ultrastructure of mitochondria, and their dysfunction results in excessive production of reactive oxygen species and release of cytochrome c. These data suggest the initiation of the mitochondrial pathway of apoptosis. Simultaneously, we observed inhibition of cell proliferation and changes in morphology of the epithelial cells toward fibroblast-like appearance, which could indicate induction of epithelial-mesenchymal transition. Thus, mitochondria are the main potential target for rifampicin in cells of epithelial origin. We suggest that similar mechanisms of pathological changes can be induced in vivo in organs and tissues accumulating rifampicin during chemotherapy of bacterial infectious diseases.

Establishment and characterization of a novel uterine carcinosarcoma cell line, TU-ECS-1, with mutations of TP53 and KRAS.

PubMed

Chiba, Yohei; Sato, Seiya; Itamochi, Hiroaki; Suga, Yasuko; Fukagawa, Tomoyuki; Oumi, Nao; Oishi, Tetsuro; Harada, Tasuku; Sugai, Tamotsu; Sugiyama, Toru

2017-04-01

A new human uterine carcinosarcoma (UCS) cell line, TU-ECS-1, was established and characterized. The morphological appearance of the cultured cells was an insular of epithelial-like cells arranged in the form of a jigsaw puzzle and mesenchymal-like cells with a spindle-shaped or fibroblast-like morphology. A relatively high proliferation rate was observed with a doubling time of 18.2 h. The chromosome number ranged from 44 to 49 and had an extra chromosome 12 (trisomy 12). The respective half-maximal inhibitory concentrations of cisplatin, paclitaxel, and doxorubicin were 2.9 µM, 154 nM, and 219 ng/mL, respectively. Mutational analysis revealed that TU-ECS-1 cells have mutations of TP53 in exons 4, 6, and 8 and of KRAS at codon 12 (G12D) in exon 2, which is a mutation hot spot on this gene. Western blot analysis showed that p53 protein was overexpressed in TU-ECS-1 cells. Immunostaining of the cultured cells and in vivo tumors showed that the TU-ECS-1 cells and xenografts were positive for epithelial marker cytokeratin AE1/3 and mesenchymal marker vimentin. These results suggested that TU-ECS-1 cells might have both epithelial and mesenchymal characteristics. This cell line may be useful to study the carcinogenesis of UCS and contribute to the development of novel treatment strategies.

Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.

1990-11-01

For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and thatmore » a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs.« less

Chromosomal changes in cultured human epithelial cells transformed by low- and high-let radiation

NASA Astrophysics Data System (ADS)

Chui-Hsu Yang, Tracy; Craise, Laurie M.; Prioleau, John C.; Stampfer, Martha R.; Rhim, Johng S.

1992-07-01

For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude mice. Neoplastic transformation was achieved by irradiating cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level.

Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

NASA Technical Reports Server (NTRS)

Craise, L. M.; Prioleau, J. C.; Stampfer, M. R.; Rhim, J. S.; Yang, TC-H (Principal Investigator)

1992-01-01

For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude mice. Neoplastic transformation was achieved by irradiating cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level.

Inhibition of PTP1B disrupts cell-cell adhesion and induces anoikis in breast epithelial cells.

PubMed

Hilmarsdottir, Bylgja; Briem, Eirikur; Halldorsson, Skarphedinn; Kricker, Jennifer; Ingthorsson, Sævar; Gustafsdottir, Sigrun; Mælandsmo, Gunhild M; Magnusson, Magnus K; Gudjonsson, Thorarinn

2017-05-11

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.

The APC tumor suppressor is required for epithelial cell polarization and three-dimensional morphogenesis

PubMed Central

Lesko, Alyssa C.; Goss, Kathleen H.; Yang, Frank F.; Schwertner, Adam; Hulur, Imge; Onel, Kenan; Prosperi, Jenifer R.

2015-01-01

The Adenomatous Polyposis Coli (APC) tumor suppressor has been previously implicated in the control of apical-basal polarity; yet, the consequence of APC loss-of-function in epithelial polarization and morphogenesis has not been characterized. To test the hypothesis that APC is required for the establishment of normal epithelial polarity and morphogenesis programs, we generated APC-knockdown epithelial cell lines. APC depletion resulted in loss of polarity and multi-layering on permeable supports, and enlarged, filled spheroids with disrupted polarity in 3D culture. Importantly, these effects of APC knockdown were independent of Wnt/β-catenin signaling, but were rescued with either full-length or a carboxy (c)-terminal segment of APC. Moreover, we identified a gene expression signature associated with APC knockdown that points to several candidates known to regulate cell-cell and cell-matrix communication. Analysis of epithelial tissues from mice and humans carrying heterozygous APC mutations further support the importance of APC as a regulator of epithelial behavior and tissue architecture. These data also suggest that the initiation of epithelial-derived tumors as a result of APC mutation or gene silencing may be driven by loss of polarity and dysmorphogenesis. PMID:25578398

Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage.

PubMed

Kanaya, Takashi; Miyazawa, Kohtaro; Takakura, Ikuro; Itani, Wataru; Watanabe, Kouichi; Ohwada, Shyuichi; Kitazawa, Haruki; Rose, Michael T; McConochie, Huw R; Okano, Hideyuki; Yamaguchi, Takahiro; Aso, Hisashi

2008-08-01

M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway.

PubMed

Ishibashi, Osamu; Akagi, Ichiro; Ogawa, Yota; Inui, Takashi

2018-05-11

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests that dysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway in esophageal squamous cell carcinoma (ESCC). To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarray-based integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell lines than the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of protein marker expression, tumorigenicity, and doxorubicin chemoresistance in two new canine mammary tumor cell lines.

PubMed

Hsiao, Yen-Ling; Hsieh, Tai-Zu; Liou, Chian-Jiun; Cheng, Yeong-Hsiang; Lin, Chung-Tien; Chang, Chi-Yao; Lai, Yu-Shen

2014-09-30

Canine mammary tumors (CMTs) are the most common type of cancer found in female dogs. Establishment and evaluation of tumor cell lines can facilitate investigations of the biological mechanisms of cancer. Different cell models are used to investigate genetic, epigenetic, and cellular pathways, cancer progression, and cancer therapeutics. Establishment of new cell models will greatly facilitate research in this field. In the present study, we established and characterized two new CMT cell lines derived from a single CMT. We established two cell lines from a single malignant CMT specimen: DTK-E and DTK-SME. Morphologically, the DTK-E cells were large, flat, and epithelial-like, whereas DTK-SME cells were round and epithelial-like. Doubling times were 24 h for DTK-E and 18 h for DTK-SME. On western blots, both cell lines expressed cytokeratin AE1, vimentin, cytokeratin 7 (CK7), and heat shock protein 27 (HSP27). Moreover, investigation of chemoresistance revealed that DTK-SME was more resistant to doxorubicin-induced apoptosis than DTK-E was. After xenotransplantation, both DTK-E and DTK-SME tumors appeared within 14 days, but the average size of DTK-SME tumors was greater than that of DTK-E tumors after 56 days. We established two new cell lines from a single CMT, which exhibit significant diversity in cell morphology, protein marker expression, tumorigenicity, and chemoresistance. The results of this study revealed that the DTK-SME cell line was more resistant to doxorubicin-induced apoptosis and exhibited higher tumorigenicity in vivo than the DTK-E cell line. We anticipate that the two novel CMT cell lines established in this study will be useful for investigating the tumorigenesis of mammary carcinomas and for screening anticancer drugs.

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

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Bradykinin-stimulated cyclooxygenase activity stimulates vas deferens epithelial anion secretion in vitro in swine and humans.

PubMed

Pierucci-Alves, Fernando; Schultz, Bruce D

2008-09-01

Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (I SC; indicating net anion secretion), an effect that was 60%-93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg9-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2 transcript is 20 times more abundant than its PTGS1 counterpart in cultured porcine vas deferens epithelia and that BDKRB2 mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase I SC in a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in I SC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion.

Bradykinin-Stimulated Cyclooxygenase Activity Stimulates Vas Deferens Epithelial Anion Secretion In Vitro in Swine and Humans1

PubMed Central

Pierucci-Alves, Fernando; Schultz, Bruce D.

2008-01-01

Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (ISC; indicating net anion secretion), an effect that was 60%–93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg9-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2 transcript is 20 times more abundant than its PTGS1 counterpart in cultured porcine vas deferens epithelia and that BDKRB2 mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase ISC in a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in ISC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion. PMID:18480467

Squamous cell carcinoma of the anal sac in five dogs.

PubMed

Esplin, D G; Wilson, S R; Hullinger, G A

2003-05-01

Tumors of the perianal area of dogs are common and include multiple tumor types. Whereas perianal adenomas occur often, adenocarcinomas of the apocrine glands of the anal sac occur less frequently. A review of the literature revealed no reports of squamous cell carcinomas arising from the epithelial lining of the anal sac. Squamous cell carcinomas originating from the lining of the anal sac were diagnosed in five dogs. Microscopically, the tumors consisted of variably sized invasive nests and cords of epithelial cells displaying squamous differentiation. Four of the five dogs were euthanatized because of problems associated with local infiltration by the tumors. In the fifth dog, there was no evidence of tumor 7 months after surgical removal, but further follow up was not available.

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

PubMed Central

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

2015-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2015-06-01

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

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

PubMed

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

2015-06-12

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

Glucocorticoid action in human corneal epithelial cells establishes roles for corticosteroids in wound healing and barrier function of the eye.

PubMed

Kadmiel, Mahita; Janoshazi, Agnes; Xu, Xiaojiang; Cidlowski, John A

2016-11-01

Glucocorticoids play diverse roles in almost all physiological systems of the body, including both anti-inflammatory and immunosuppressive roles. Synthetic glucocorticoids are one of the most widely prescribed drugs and are used in the treatment of conditions such as autoimmune diseases, allergies, ocular disorders and certain types of cancers. In the interest of investigating glucocorticoid actions in the cornea of the eye, we established that multiple cell types in mouse corneas express functional glucocorticoid receptor (GR) with corneal epithelial cells having robust expression. To define glucocorticoid actions in a cell type-specific manner, we employed immortalized human corneal epithelial (HCE) cell line to define the glucocorticoid transcriptome and elucidated its functions in corneal epithelial cells. Over 4000 genes were significantly regulated within 6 h of dexamethasone treatment, and genes associated with cell movement, cytoskeletal remodeling and permeability were highly regulated. Real-time in vitro wound healing assays revealed that glucocorticoids delay wound healing by attenuating cell migration. These functional alterations were associated with cytoskeletal remodeling at the wounded edge of a scratch-wounded monolayer. However, glucocorticoid treatment improved the organization of tight-junction proteins and enhanced the epithelial barrier function. Our results demonstrate that glucocorticoids profoundly alter corneal epithelial gene expression and many of these changes likely impact both wound healing and epithelial cell barrier function. Published by Elsevier Ltd.

Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program.

PubMed

Lamorte, Louie; Rodrigues, Sonia; Naujokas, Monica; Park, Morag

2002-10-04

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

Curcumin suppresses AGEs induced apoptosis in tubular epithelial cells via protective autophagy

PubMed Central

Wei, Ying; Gao, Jiaqi; Qin, Lingling; Xu, Yunling; Shi, Haoxia; Qu, Lingxia; Liu, Yongqiao; Xu, Tunhai; Liu, Tonghua

2017-01-01

Renal tubular cell apoptosis and tubular dysfunction is an important process underlying diabetic nephropathy (DN). Understanding the mechanisms underlying renal tubular epithelial cell survival is important for the prevention of kidney damage associated with glucotoxicity. Curcumin has been demonstrated to possess potent anti-apoptotic properties. However, the roles of curcumin in renal epithelial cells are yet to be defined. The present study investigated advanced glycation or glycoxidation end-product (AGE)-induced toxicity in renal tubular epithelial cells via several complementary assays, including cell viability, cell apoptosis and cell autophagy in the NRK-52E rat kidney tubular epithelial cell line. The extent of apoptosis was significantly increased in the NRK-52E cells following treatment with AGEs. The results also indicated that curcumin reversed this effect by promoting autophagy through the phosphoinositide 3-kinase/AKT serine/threonine kinase signaling pathway. These conclusions suggested that curcumin exerts a renoprotective effect in the presence of AGEs, at least in part by activating autophagy in NRK-52E cells. Collectively, these findings indicate that curcumin not only exerts renoprotective effects, however may also act as a novel therapeutic strategy for the treatment of diabetic nephropathy. PMID:29285156

Inhibition of PTP1B disrupts cell–cell adhesion and induces anoikis in breast epithelial cells

PubMed Central

Hilmarsdottir, Bylgja; Briem, Eirikur; Halldorsson, Skarphedinn; Kricker, Jennifer; Ingthorsson, Sævar; Gustafsdottir, Sigrun; Mælandsmo, Gunhild M; Magnusson, Magnus K; Gudjonsson, Thorarinn

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell–cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype. PMID:28492548

[Establishment of Z-HL16C cell line.].

PubMed

Chen, J P; Li, J; Zhao, S L; Tian, J Y; Ye, F

2006-09-01

To establish and study the nature and the application of Z-HL16C cell line. The cell line was continuously passed, frozen stored and recovered. Its application was expanded and the cell type was identified. The cell line had an epithelial-cell-like shape, the size appeared uniform, the cell boundary was distinct. It has been continuously passed, frozen stored and recovered for ten years. Its recovery rate was about 90%. It has been proved to be sensitive to the tested viruses which were enteroviruses (Polio, Cox, Echo), influenza viruses, parainfluenzaviruses, adenoviruses, measles virus. This cell line has been identified as a cancerization cell. The cell line Z-HL16C has been stably established, it has a broad spectrum in sensitivity for culturing viruses.

Involvement of CRF2 signaling in enterocyte differentiation

PubMed Central

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-01-01

AIM To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. RESULTS CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. CONCLUSION Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases. PMID:28811708

Involvement of CRF2 signaling in enterocyte differentiation.

PubMed

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-07-28

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs).

PubMed

Shi, Huaiping; Shi, Hengbo; Luo, Jun; Wang, Wei; Haile, Abiel B; Xu, Huifen; Li, Jun

2014-07-01

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post-parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT-MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF-7 cells. hT-MECs on passage 42 grew vigorously and 'S' sigmoid curves of growth were observed. Moreover, hT-MECs maintained a normal chromosome modal number of 2n=60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta-casein protein was synthesized and secreted. Beta-casein expression was enhanced by prolactin (P<0.05). Lipid droplets were found in hT-MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT-MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time. © 2014 Japanese Society of Animal Science.

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity.

PubMed

Jayachandran, Aparna; Shrestha, Ritu; Dhungel, Bijay; Huang, I-Tao; Vasconcelos, Marianna Yumi Kawashima; Morrison, Brian J; Ramlogan-Steel, Charmaine A; Steel, Jason C

2017-09-26

To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai , Zeb and Twist family of EMT transcription factors. Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

PubMed

Carmody, Leigh C; Germain, Andrew R; VerPlank, Lynn; Nag, Partha P; Muñoz, Benito; Perez, Jose R; Palmer, Michelle A J

2012-10-01

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

A chicken influenza virus recognizes fucosylated α2,3 sialoglycan receptors on the epithelial cells lining upper respiratory tracts of chickens.

PubMed

Hiono, Takahiro; Okamatsu, Masatoshi; Nishihara, Shoko; Takase-Yoden, Sayaka; Sakoda, Yoshihiro; Kida, Hiroshi

2014-05-01

Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SAα2,3Gal glycans were detected, but not linear SAα2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SAα2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucose-branched SAα2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR. Copyright © 2014 Elsevier Inc. All rights reserved.

Spontaneous Transformation of Murine Epithelial Cells Requires the Early Acquisition of Specific Chromosomal Aneuploidies and Genomic Imbalances

PubMed Central

Padilla-Nash, Hesed M.; Hathcock, Karen; McNeil, Nicole E.; Mack, David; Hoeppner, Daniel; Ravin, Rea; Knutsen, Turid; Yonescu, Raluca; Wangsa, Danny; Dorritie, Kathleen; Barenboim, Linda; Hu, Yue; Ried, Thomas

2011-01-01

Human carcinomas are defined by recurrent chromosomal aneuploidies, which result in tissue-specific distribution of genomic imbalances. In order to develop models for these genome mutations and determine their role in tumorigenesis, we generated 45 spontaneously transformed murine cell lines from normal epithelial cells derived from bladder, cervix, colon, kidney, lung, and mammary gland. Phenotypic changes, chromosomal aberrations, centrosome number, and telomerase activity were assayed in control uncultured cells and in three subsequent stages of transformation. Supernumerary centrosomes, bi-nucleate cells, and tetraploidy were observed as early as 48 hr after explantation. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, promoted genome duplication. Spectral karyotyping demonstrated that aneuploidy preceded immortalization, consisting predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 10, 15, and 19). After transformation, focal amplifications of the oncogenes Myc and Mdm2 were frequently detected. Fifty percent of the transformed lines resulted in tumors upon injection into immuno-compromised mice. The phenotypic and genomic alterations observed in spontaneously transformed murine epithelial cells recapitulated the aberration pattern observed during human carcinogenesis. The dominant aberration of these cell lines was the presence of specific chromosomal aneuploidies. We propose that our newly derived cancer models will be useful tools to dissect the sequential steps of genome mutations during malignant transformation, and also to identify cancer-specific genes, signaling pathways, and the role of chromosomal instability in this process. PMID:22161874

Generation of organotypic raft cultures from primary human keratinocytes.

PubMed

Anacker, Daniel; Moody, Cary

2012-02-22

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)(1). The life cycle of HPV is tightly linked to the differentiation of squamous epithelium(2). Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production(3,4,5). In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras(6) and modified by Kopan et al.(7), the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies(8). Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses(9). Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

Multi-resolution cell orientation congruence descriptors for epithelium segmentation in endometrial histology images.

PubMed

Li, Guannan; Raza, Shan E Ahmed; Rajpoot, Nasir M

2017-04-01

It has been recently shown that recurrent miscarriage can be caused by abnormally high ratio of number of uterine natural killer (UNK) cells to the number of stromal cells in human female uterus lining. Due to high workload, the counting of UNK and stromal cells needs to be automated using computer algorithms. However, stromal cells are very similar in appearance to epithelial cells which must be excluded in the counting process. To exclude the epithelial cells from the counting process it is necessary to identify epithelial regions. There are two types of epithelial layers that can be encountered in the endometrium: luminal epithelium and glandular epithelium. To the best of our knowledge, there is no existing method that addresses the segmentation of both types of epithelium simultaneously in endometrial histology images. In this paper, we propose a multi-resolution Cell Orientation Congruence (COCo) descriptor which exploits the fact that neighbouring epithelial cells exhibit similarity in terms of their orientations. Our experimental results show that the proposed descriptors yield accurate results in simultaneously segmenting both luminal and glandular epithelium. Copyright © 2017 Elsevier B.V. All rights reserved.

Platelets are a possible regulator of human endometrial re-epithelialization during menstruation.

PubMed

Suginami, Koh; Sato, Yukiyasu; Horie, Akihito; Matsumoto, Hisanori; Kyo, Satoru; Araki, Yoshihiko; Konishi, Ikuo; Fujiwara, Hiroshi

2017-01-01

The human endometrium periodically breaks down and regenerates. As platelets have been reported to contribute to the tissue remodeling process, we examined the possible involvement of platelets in endometrial regeneration. The distribution of extravasating platelets throughout the menstrual cycle was immunohistochemically examined using human endometrial tissues. EM-E6/E7/hTERT cells, a human endometrial epithelial cell-derived immortalized cell line, were co-cultured with platelets, and the effects of platelets on the epithelialization response of EM-E6/E7/hTERT cells were investigated by attachment and permeability assays, immunohistochemical staining, and Western blot analysis. Immunohistochemical study showed numerous extravasated platelets in the subluminar stroma during the menstrual phase. The platelets promoted the cell-to-matrigel attachment of EM-E6/E7/hTERT cells concomitantly with the phosphorylation of focal adhesion kinase. They also promoted cell-to-cell contact among EM-E6/E7/hTERT cells in parallel with E-cadherin expression. These results indicate the possible involvement of platelets in the endometrial epithelial re-epithelialization process. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Availability of a fetal goat tongue cell line ZZ-R 127 for isolation of Foot-and-mouth disease virus (FMDV) from clinical samples collected from animals experimentally infected with FMDV.

PubMed

Fukai, Katsuhiko; Onozato, Hiroyuki; Kitano, Rie; Yamazoe, Reiko; Morioka, Kazuki; Yamada, Manabu; Ohashi, Seiichi; Yoshida, Kazuo; Kanno, Toru

2013-11-01

The availability of the fetal goat tongue cell line ZZ-R 127 for the isolation of Foot-and-mouth disease virus (FMDV) has not been evaluated using clinical samples other than epithelial suspensions. Therefore, in the current study, the availability of ZZ-R 127 cells for the isolation of FMDV was evaluated using clinical samples (e.g., sera, nasal swabs, saliva, feces, and oropharyngeal fluids) collected from animals experimentally infected with an FMDV isolate. Virus isolation rates for the ZZ-R 127 cells were statistically higher than those for the porcine kidney cell line (IB-RS-2) in experimental infections using cattle, goats, and pigs (P < 0.01). Virus titers in the ZZ-R 127 cells were also statistically higher than those in the IB-RS-2 cells. The availability of ZZ-R 127 cells for the isolation of FMDV not only from epithelial suspensions but also from other clinical samples was confirmed in the current study.

Activation of Neurokinin-1 Receptors during Ozone Inhalation Contributes to Epithelial Injury and Repair

PubMed Central

Oslund, Karen L.; Hyde, Dallas M.; Putney, Leialoha F.; Alfaro, Mario F.; Walby, William F.; Tyler, Nancy K.; Schelegle, Edward S.

2008-01-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2′-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2′-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress–positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways. PMID:18390473

Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential

NASA Technical Reports Server (NTRS)

Piao, C. Q.; Willey, J. C.; Hei, T. K.; Hall, E. J. (Principal Investigator)

1999-01-01

The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.

A Riboproteomic Platform to Identify Novel Targets for Prostate Cancer Therapy

DTIC Science & Technology

2015-10-01

cell lines derived from RWPE1 prostatic epithelial cells after exposure to N-methyl-N- nitrosourea (MNU) (these cell lines are commercially available...is well established that the malignancy of cells is strongly linked to and dependent on aberrant protein synthesis . Current knowledge clearly...highlights deregulation of protein synthesis , in the development of prostate cancer, through aberrant activation of classical signaling pathways. It has

CLOCK regulates mammary epithelial cell growth and differentiation

PubMed Central

Crodian, Jennifer; Suárez-Trujillo, Aridany; Erickson, Emily; Weldon, Bethany; Crow, Kristi; Cummings, Shelby; Chen, Yulu; Shamay, Avi; Mabjeesh, Sameer J.; Plaut, Karen

2016-01-01

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63. qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock. These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland. PMID:27707717

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions

DOE PAGES

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.; ...

2015-07-30

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Activation of calcitonin gene-related peptide receptor during ozone inhalation contributes to airway epithelial injury and repair.

PubMed

Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

2009-10-01

The authors investigated the importance of the neuropeptide, calcitonin gene-related peptide (CGRP), in epithelial injury, repair, and neutrophil emigration after ozone exposure. Wistar rats were administered either a CGRP-receptor antagonist (CGRP(8-37)) or saline and exposed to 8 hours of 1-ppm ozone or filtered air with an 8-hour postexposure period. Immediately after exposure, ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, airway dissected lung lobes were stained for 5'-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Positive epithelial cells were quantified in specific airway generations. Rats treated with CGRP(8-37) had significantly reduced epithelial injury in terminal bronchioles and reduced epithelial proliferation in proximal airways and terminal bronchioles. Bronchoalveolar lavage and sections of terminal bronchioles showed no significant difference in the number of neutrophils emigrating into airways in CGRP(8-37)-treated rats. The airway epithelial cell line, HBE-1, showed no difference in the number of oxidant stress positive cells during exposure to hydrogen peroxide and a range of CGRP(8-37) doses, demonstrating no antioxidant effect of CGRP(8-37). We conclude that activation of CGRP receptors during ozone inhalation contributes to airway epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

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

PubMed Central

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

2014-01-01

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

Different molecular organization of two carotenoids, lutein and zeaxanthin, in human colon epithelial cells and colon adenocarcinoma cells

NASA Astrophysics Data System (ADS)

Grudzinski, Wojciech; Piet, Mateusz; Luchowski, Rafal; Reszczynska, Emilia; Welc, Renata; Paduch, Roman; Gruszecki, Wieslaw I.

2018-01-01

Two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of exogenous carotenoids, either zeaxanthin or lutein. Both carotenoids demonstrated cytotoxicity with respect to cancer cells but not to normal cells. Cells from both the cell lines were analyzed with application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. Both imaging techniques show effective incorporation of carotenoid molecules into growing cells. Comparison of the Raman scattering and fluorescence lifetime characteristics reveals different molecular organization of carotenoids in the carcinoma and normal cells. The main difference consists in a carotenoid aggregation level which is substantially lower in the carcinoma cells as compared to the normal cells. Different molecular organization of carotenoids was interpreted in terms of a different metabolism of normal and carcinoma cells and has been concluded to provide a possibility of cancer diagnosis based on spectroscopic analyses.

Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

PubMed

Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

2018-02-01

Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

Inhibitory effect of green tea on injury to a cultured renal epithelial cell line, LLC-PK1.

PubMed

Yokozawa, T; Dong, E; Chung, H Y; Oura, H; Nakagawa, H

1997-01-01

When cells from a cultured renal epithelial cell line, LLC-PK1, were cultured under hypoxic conditions (oxygen concentration of 2% or less) before reoxygenation was applied (95% air, 5% CO2), the leakage of lactate dehydrogenase (LDH) into the medium increased. This phenomenon was inhibited in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, suggesting the involvement of free radicals. Such oxidative stress was significantly inhibited by a green tea extract, and more potently by a tannin mixture. On the other hand, under ordinary culture conditions (95%, air, 5% CO2), there was cell injury, although the LDH leakage was less than that under hypoxia/reoxygenation, and such injury was inhibited by the green tea extract and the tannin mixture.

Gene amplification and microsatellite instability induced in tumorigenic human bronchial epithelial cells by alpha particles and heavy ions

NASA Technical Reports Server (NTRS)

Piao, C. Q.; Hei, T. K.; Hall, E. J. (Principal Investigator)

2001-01-01

Gene amplification and microsatellite alteration are useful markers of genomic instability in tumor and transformed cell lines. It has been suggested that genomic instability contributes to the progression of tumorigenesis by accumulating genetic changes. In this study, amplification of the carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) gene in transformed and tumorigenic human bronchial epithelial (BEP2D) cells induced by either alpha particles or (56)Fe ions was assessed by measuring resistance to N-(phosphonacetyl)-l-aspartate (PALA). In addition, alterations of microsatellite loci located on chromosomes 3p and 18q were analyzed in a series of primary and secondary tumor cell lines generated in nude mice. The frequency of PALA-resistant colonies was 1-3 x 10(-3) in tumor cell lines, 5-8 x 10(-5) in transformed cells prior to inoculation into nude mice, and less than 10(-7) in control BEP2D cells. Microsatellite alterations were detected in all 11 tumor cell lines examined at the following loci: D18S34, D18S363, D18S877, D3S1038 and D3S1607. No significant difference in either PALA resistance or microsatellite instability was found in tumor cell lines that were induced by alpha particles compared to those induced by (56)Fe ions.

Isolation and Growth of Prostate Stem Cells and Establishing Cancer Cell Lines from Human Prostate Tumors

DTIC Science & Technology

2009-05-01

contaminating rat UGSE cells ; and regions of host mouse glands were either from circulating pluripotent stem cells or local epithelial cells which were...CONTRACT NUMBER Isolation and Growth of Prostate Stem Cells and Establishing Cancer Cell Lines from Human Prostate Tumors 5b. GRANT NUMBER 81WXH...NOTES 14. ABSTRACT The objective of this proposal was to isolate, grow, and characterize normal prostate stem cells and establish new prostate

Establishment and characterization of fin-derived cell line from ornamental carp, Cyprinus carpio koi, for virus isolation in India.

PubMed

Swaminathan, T Raja; Basheer, V S; Kumar, Raj; Kathirvelpandian, A; Sood, Neeraj; Jena, J K

2015-08-01

Cyprinus carpio koi fin (CCKF) cell line was established and characterized from the caudal fin tissue of ornamental common carp, C. carpio koi. This cell line has been maintained in L-15 medium supplemented with 15% foetal bovine serum (FBS) and subcultured more than 52 times over a period of 24 mo. The CCKF cell line consisted of epithelial cells and was able to grow at temperatures between 22 and 35°C with an optimum temperature of 28°C. The growth rate of these cells increased as the proportion of FBS increased from 2 to 20% with optimum growth at the concentrations of 15% FBS. Karyotype analysis revealed that the modal chromosome number of CCKF cells was 2n = 100. Partial amplification and sequencing of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CCKF cell line originated from ornamental common carp. The CCKF cells showed strong reaction to the cytokeratin marker, indicating that it was epithelial in nature. The extracellular products of Vibrio cholerae MTCC 3904 and Aeromonas hydrophila were toxic to the CCKF cells and not susceptible to viral nervous necrosis virus (VNNV). These CCKF cells were confirmed for the absence of Mycoplasma sp. by polymerase chain reaction. Furthermore, 90% of viable cells could be effectively revived 4 mo after cryopreservation from CCKF cell population suggesting the possibility of long-term storage of the cells.

Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xiangjun; Yao, Qisheng, E-mail: yymcyqs@126.com; Sun, Xinbo

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treatedmore » with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway. - Highlights: • Slit2 ameliorates inflammation after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates fibrosis after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates inflammation and fibrosis after hypoxia-and LPS-induced renal epithelial cells injury via TLR4/NF-κB.« less

Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function.

PubMed

Rosenberg, I M; Göke, M; Kanai, M; Reinecker, H C; Podolsky, D K

1997-10-01

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

Innate immunity in HIV-1 infection: epithelial and non-specific host factors of mucosal immunity- a workshop report.

PubMed

Nittayananta, W; Weinberg, A; Malamud, D; Moyes, D; Webster-Cyriaque, J; Ghosh, S

2016-04-01

The interplay between HIV-1 and epithelial cells represents a critical aspect in mucosal HIV-1 transmission. Epithelial cells lining the oral cavity cover subepithelial tissues, which contain virus-susceptible host cells including CD4(+) T lymphocytes, monocytes/macrophages, and dendritic cells. Oral epithelia are among the sites of first exposure to both cell-free and cell-associated virus HIV-1 through breast-feeding and oral-genital contact. However, oral mucosa is considered to be naturally resistant to HIV-1 transmission. Oral epithelial cells have been shown to play a crucial role in innate host defense. Nevertheless, it is not clear to what degree these local innate immune factors contribute to HIV-1 resistance of the oral mucosa. This review paper addressed the following issues that were discussed at the 7th World Workshop on Oral Health and Disease in AIDS held in Hyderabad, India, during November 6-9, 2014: (i) What is the fate of HIV-1 after interactions with oral epithelial cells?; (ii) What are the keratinocyte and other anti-HIV effector oral factors, and how do they contribute to mucosal protection?; (iii) How can HIV-1 interactions with oral epithelium affect activation and populations of local immune cells?; (iv) How can HIV-1 interactions alter functions of oral epithelial cells? © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Significance of CD133 positive cells in four novel HPV-16 positive cervical cancer-derived cell lines and biopsies of invasive cervical cancer.

PubMed

Javed, Shifa; Sharma, Bal Krishan; Sood, Swati; Sharma, Sanjeev; Bagga, Rashmi; Bhattacharyya, Shalmoli; Rayat, Charan Singh; Dhaliwal, Lakhbir; Srinivasan, Radhika

2018-04-02

Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important. Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed. Four low-passage novel cell lines designated RSBS-9, - 14 and - 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133 + phenotype. In tissue samples of untreated invasive cervical cancer, CD133 + CSCs ranged from 1.3-23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133 + with CD133 - bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity. Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133 + phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.

Osthole inhibits the tumorigenesis of hepatocellular carcinoma cells.

PubMed

Lin, Zhi-Kun; Liu, Jia; Jiang, Guo-Qiang; Tan, Guang; Gong, Peng; Luo, Hai-Feng; Li, Hui-Min; Du, Jian; Ning, Zhen; Xin, Yi; Wang, Zhong-Yu

2017-03-01

Hepatocellular carcinoma (HCC) accounts for approximately 90% of all cases of primary liver cancer, and the majority of patients with HCC are deprived of effective curative methods. Osthole is a Chinese herbal medicine which has been reported to possess various pharmacological functions, including hepatocellular protection. In the present study, we investigated the anticancer activity of osthole using HCC cell lines. We found that osthole inhibited HCC cell proliferation, induced cell cycle arrest, triggered DNA damage and suppressed migration in HCC cell lines. Furthermore, we demonstrated that osthole not only contributed to cell cycle G2/M phase arrest via downregulation of Cdc2 and cyclin B1 levels, but also induced DNA damage via an increase in ERCC1 expression. In addition, osthole inhibited the migration of HCC cell lines by significantly downregulating MMP-2 and MMP-9 levels. Finally, we demonstrated that osthole inhibited epithelial-mesenchymal transition (EMT) via increasing the expression of epithelial biomarkers E-cadherin and β-catenin, and significantly decreasing mesenchymal N-cadherin and vimentin protein expression. These results suggest that osthole may have potential chemotherapeutic activity against HCC.

The Development of M Cells in Peyer’s Patches Is Restricted to Specialized Dome-Associated Crypts

PubMed Central

Gebert, Andreas; Fassbender, Susanne; Werner, Kerstin; Weissferdt, Annikka

1999-01-01

It is controversial whether the membranous (M) cells of the Peyer’s patches represent a separate cell line or develop from enterocytes under the influence of lymphocytes on the domes. To answer this question, the crypts that produce the dome epithelial cells were studied and the distribution of M cells over the domes was determined in mice. The Ulex europaeus agglutinin was used to detect M cells in mouse Peyer’s patches. Confocal microscopy with lectin-gold labeling on ultrathin sections, scanning electron microscopy, and laminin immuno-histochemistry were combined to characterize the cellular composition and the structure of the dome-associated crypts and the dome epithelium. In addition, the sites of lymphocyte invasion into the dome epithelium were studied after removal of the epithelium using scanning electron microscopy. The domes of Peyer’s patches were supplied with epithelial cells that derived from two types of crypt: specialized dome-associated crypts and ordinary crypts differing not only in shape, size, and cellular composition but also in the presence of M cell precursors. When epithelial cells derived from ordinary crypts entered the domes, they formed converging radial strips devoid of M cells. In contrast to the M cells, the sites where lymphocytes invaded the dome epithelium were not arranged in radial strips, but randomly distributed over the domes. M cell development is restricted to specialized dome-associated crypts. Only dome epithelial cells that derive from these specialized crypts differentiate into M cells. It is concluded that M cells represent a separate cell line that is induced in the dome-associated crypts by still unknown, probably diffusible lymphoid factors. PMID:10329609

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

PubMed

Kasurinen, Stefanie; Happo, Mikko S; Rönkkö, Teemu J; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I

2018-01-01

In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion

PubMed Central

Happo, Mikko S.; Rönkkö, Teemu J.; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I.

2018-01-01

Background In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. Methods We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. Results We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. Conclusions All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity. PMID:29466392

Epithelial-mesenchymal transition in tissue repair and fibrosis.

PubMed

Stone, Rivka C; Pastar, Irena; Ojeh, Nkemcho; Chen, Vivien; Liu, Sophia; Garzon, Karen I; Tomic-Canic, Marjana

2016-09-01

The epithelial-mesenchymal transition (EMT) describes the global process by which stationary epithelial cells undergo phenotypic changes, including the loss of cell-cell adhesion and apical-basal polarity, and acquire mesenchymal characteristics that confer migratory capacity. EMT and its converse, MET (mesenchymal-epithelial transition), are integral stages of many physiologic processes and, as such, are tightly coordinated by a host of molecular regulators. Converging lines of evidence have identified EMT as a component of cutaneous wound healing, during which otherwise stationary keratinocytes (the resident skin epithelial cells) migrate across the wound bed to restore the epidermal barrier. Moreover, EMT plays a role in the development of scarring and fibrosis, as the matrix-producing myofibroblasts arise from cells of the epithelial lineage in response to injury but are pathologically sustained instead of undergoing MET or apoptosis. In this review, we summarize the role of EMT in physiologic repair and pathologic fibrosis of tissues and organs. We conclude that further investigation into the contribution of EMT to the faulty repair of fibrotic wounds might identify components of EMT signaling as common therapeutic targets for impaired healing in many tissues. Graphical Abstract Model for injury-triggered EMT activation in physiologic wound repair (left) and fibrotic wound healing (right).

Amphiregulin suppresses epithelial cell apoptosis in lipopolysaccharide-induced lung injury in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogata-Suetsugu, Saiko; Yanagihara, Toyoshi; Hamada, Naoki

Background and objective: As a member of the epidermal growth factor family, amphiregulin contributes to the regulation of cell proliferation. Amphiregulin was reported to be upregulated in damaged lung tissues in patients with chronic obstructive pulmonary disease and asthma and in lung epithelial cells in a ventilator-associated lung injury model. In this study, we investigated the effect of amphiregulin on lipopolysaccharide (LPS)-induced acute lung injury in mice. Methods: Acute lung injury was induced by intranasal instillation of LPS in female C57BL/6 mice, and the mice were given intraperitoneal injections of recombinant amphiregulin or phosphate-buffered saline 6 and 0.5 h before andmore » 3 h after LPS instillation. The effect of amphiregulin on apoptosis and apoptotic pathways in a murine lung alveolar type II epithelial cell line (LA-4 cells) were examined using flow cytometry and western blotting, respectively. Results: Recombinant amphiregulin suppressed epithelial cell apoptosis in LPS-induced lung injury in mice. Western blotting revealed that amphiregulin suppressed epithelial cell apoptosis by inhibiting caspase-8 activity. Conclusion: Amphiregulin signaling may be a therapeutic target for LPS-induced lung injury treatment through its prevention of epithelial cell apoptosis. - Highlights: • Amphiregulin suppresses epithelial cell apoptosis in LPS-induced lung injury in mice. • The mechanism relies on inhibiting caspase-8 activity. • Amphiregulin signaling may be a therapeutic target for LPS-induced lung injury.« less

Dysregulation of Lysyl Oxidase Expression in Lesions and Endometrium of Women With Endometriosis

PubMed Central

Ruiz, Lynnette A.; Báez-Vega, Perla M.; Ruiz, Abigail; Peterse, Daniëlle P.; Monteiro, Janice B.; Bracero, Nabal; Beauchamp, Pedro; Fazleabas, Asgerally T.; Flores, Idhaliz

2015-01-01

Lysyl oxidases (LOXs) are enzymes involved in collagen deposition, extracellular membrane remodeling, and invasive/metastatic potential. Previous studies reveal an association of LOXs and endometriosis. We aimed to identify the mechanisms activated by upregulation of lysyl oxidases (LOX) in endometriotic cells and tissues. We hypothesized that LOX plays a role in endometriosis by promoting invasiveness and epithelial to mesenchymal transition (EMT). Methods: The LOX protein expression levels were measured by immunohistochemistry in lesions and endometrium on a tissue microarray (TMA) and in endometrial biopsies from patients and controls during the window of implantation (WOI). Estradiol regulation of LOX expression was determined by quantitative polymerase chain reaction (qPCR). Proliferation, invasion, and migration assays were performed in epithelial (endometrial epithelial cell), endometrial (human endometrial stromal cell), and endometriotic cell lines (ECL and 12Z). Pathway-focused multiplex qPCR was used to determine transcriptome changes due to LOX overexpression. Results: LOX protein was differentially expressed in ovarian versus peritoneal lesions. During WOI, LOX levels were higher in luminal epithelium of patients with endometriosis-associated infertility compared to controls. Invasive epithelial cell lines expressed higher levels of LOX than noninvasive ones. Transfection of LOX into noninvasive epithelial cells increased their migration in an LOX inhibitor-sensitive manner. Overexpression of LOX did not fully induce EMT but the expression of genes related to fibrosis and extracellular matrix remodeling were dysregulated. Conclusions: This study documents that expression of LOX is differentially regulated in endometriotic lesions and endometrium. A role for LOX in mediating proliferation, migration, and invasion of endometrial and endometriotic cells was observed, which may be implicated in the establishment and progression of endometriotic lesions. PMID:25963914

Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

PubMed Central

Ferrer-Martinez, A; Felipe, A; Nicholson, B; Casado, J; Pastor-Anglada, M; McGivan, J

1995-01-01

The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself. Images Figure 4 PMID:7654212

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

PubMed

Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

Transcriptional Responses of Candida albicans to Epithelial and Endothelial Cells▿ †

PubMed Central

Park, Hyunsook; Liu, Yaoping; Solis, Norma; Spotkov, Joshua; Hamaker, Jessica; Blankenship, Jill R.; Yeaman, Michael R.; Mitchell, Aaron P.; Liu, Haoping; Filler, Scott G.

2009-01-01

Candida albicans interacts with oral epithelial cells during oropharyngeal candidiasis and with vascular endothelial cells when it disseminates hematogenously. We set out to identify C. albicans genes that govern interactions with these host cells in vitro. The transcriptional response of C. albicans to the FaDu oral epithelial cell line and primary endothelial cells was determined by microarray analysis. Contact with epithelial cells caused a decrease in transcript levels of genes related to protein synthesis and adhesion, whereas contact with endothelial cells did not significantly influence any specific functional category of genes. Many genes whose transcripts were increased in response to either host cell had not been previously characterized. We constructed mutants with homozygous insertions in 22 of these uncharacterized genes to investigate their function during host-pathogen interaction. By this approach, we found that YCK2, VPS51, and UEC1 are required for C. albicans to cause normal damage to epithelial cells and resist antimicrobial peptides. YCK2 is also necessary for maintenance of cell polarity. VPS51 is necessary for normal vacuole formation, resistance to multiple stressors, and induction of maximal endothelial cell damage. UEC1 encodes a unique protein that is required for resistance to cell membrane stress. Therefore, some C. albicans genes whose transcripts are increased upon contact with epithelial or endothelial cells are required for the organism to damage these cells and withstand the stresses that it likely encounters during growth in the oropharynx and bloodstream. PMID:19700637

Cell Motility and Jamming across the EMT

NASA Astrophysics Data System (ADS)

Grosser, Steffen; Oswald, Linda; Lippoldt, Jürgen; Heine, Paul; Kaes, Josef A.

We use single-cell tracking and cell shape analysis to highlight the different roles that cell jamming plays in the behaviour of epithelial vs. mesenchymal mammary breast cell lines (MCF-10A, MDA-MB-231) in 2D adherent culture. An automatic segmentation allows for the evaluation of cell shapes, which we compare to predictions made by the self-propelled vertex (SPV) model. On top of that, we employ co-cultures to study the emerging demixing behaviour of these cell lines, demonstrating that the mesenchymal MDA-MB-231 cell line forms unjammed islands within the jammed collective.

The Human Polymeric Immunoglobulin Receptor Facilitates Invasion of Epithelial Cells by Streptococcus pneumoniae in a Strain-Specific and Cell Type-Specific Manner

PubMed Central

Brock, Sean C.; McGraw, Patricia A.; Wright, Peter F.; Crowe Jr., James E.

2002-01-01

Streptococcus pneumoniae is a gram-positive bacterial pathogen that causes invasive life-threatening disease worldwide. This organism also commonly colonizes the upper respiratory epithelium in an asymptomatic fashion. To invade, this pathogen must traverse the respiratory epithelial barrier, allowing it to cause disease locally or disseminate hematogenously throughout the body. Previous work has demonstrated that S. pneumoniae choline-binding protein A, a pneumococcal surface protein, interacts specifically with the human polymeric immunoglobulin receptor, which is expressed by cells in the respiratory epithelium. Choline-binding protein A is required for efficient colonization of the nasopharynx in vivo. Additionally, a recent study showed that the R6x laboratory strain of S. pneumoniae invades a human pharyngeal cell line in a human polymeric immunoglobulin receptor-dependent manner. These findings raised the possibility that the interaction between choline-binding protein A and human polymeric immunoglobulin receptor may be a key determinant of S. pneumoniae pathogenesis. However, the strain used in prior invasion studies, R6x, is an unencapsulated, nonpathogenic strain. In the present study we determined the relative ability of strain R6x or pathogenic strains to invade a variety of human polymeric immunoglobulin receptor-expressing epithelial cell lines. The results of this work suggest that human polymeric immunoglobulin receptor-dependent enhanced invasion of epithelial cells by S. pneumoniae is a limited phenomenon that occurs in a strain-specific and cell type-specific manner. PMID:12183558

Oxytetracycline Inhibits Mucus Secretion and Inflammation in Human Airway Epithelial Cells.

PubMed

Shah, Said Ahmad; Ishinaga, Hajime; Takeuchi, Kazuhiko

2017-01-01

Oxytetracycline is a broad-spectrum antibiotic, but its nonantibacterial effects in the human respiratory tract are unknown. In this study, the effects of oxytetracycline on mucus secretion and inflammation were examined by PCR and ELISA in the human airway epithelial cell line NCI-H292. Oxytetracycline (10 μg/mL) significantly inhibited TNF-α-induced MUC5AC gene expression and MUC5AC protein levels in NCI-H292 cells. It also downregulated IL-8 and IL-1β gene expression and IL-1β protein levels. Our findings demonstrated that oxytetracycline suppressed mucus production and inflammation in human respiratory epithelial cells, providing further evidence for the usefulness of oxytetracycline for human airway inflammatory diseases. © 2017 S. Karger AG, Basel.

Superhydrophobic surfaces allow probing of exosome self organization using X-ray scattering

NASA Astrophysics Data System (ADS)

Accardo, Angelo; Tirinato, Luca; Altamura, Davide; Sibillano, Teresa; Giannini, Cinzia; Riekel, Christian; di Fabrizio, Enzo

2013-02-01

Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates.Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr34032e

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins.

PubMed

Carduner, L; Leroy-Dudal, J; Picot, C R; Gallet, O; Carreiras, F; Kellouche, S

2014-08-01

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

[Establishment of goat limbal stem cell strain expressing Venus fluorescent protein and construction of limbal epithelial sheets].

PubMed

Yin, Jiqing; Liu, Wenqiang; Liu, Chao; Zhao, Guimin; Zhang, Yihua; Liu, Weishuai; Hua, Jinlian; Dou, Zhongying; Lei, Anmin

2010-12-01

The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT.

Up-Regulation of Pro-Inflammatory Cytokines and Chemokine Production in Avian Influenza H9N2 Virus-Infected Human Lung Epithelial Cell Line (A549).

PubMed

Farzin, Hamidreza; Toroghi, Reza; Haghparast, Alireza

2016-01-01

Influenza H9N2 virus mostly infects avian species but poses a potential health risk to humans. Little is known about the mammalian host immune responses to H9N2 virus. To obtain insight into the innate immune responses of human lung epithelial cells to the avian H9N2 virus, the expressions of pro-inflammatory cytokines and chemokine in the human airway epithelial cells infected with avian H9N2 virus were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). H9N2 virus was able to cultivate in the human lung epithelial cell line (A549) and stimulate production of pro-inflammatory cytokines (IL-1β, IL-6) and chemokine (IL-8). Expressions of cytokine genes were up-regulated to a significantly higher level for IL-1β (p < 0.01), IL-6 (p < 0.01 after 12 hours and p < 0.05 after 24 hours) and IL-8 (p < 0.01 after 12 hours and p < 0.001 after 24 hours) in virus-cultured A549 cells as compared with non-virus-cultured cells. The amount of IL-6 and IL-1β proteins secreted into the culture medium was also increased after virus culture infection of A549 cell line compared to non-virus-cultured A549 cells and were significant in both IL-1β (p < 0.05 in 18 hours and p < 0.001 in 24-48 hours harvested supernatant) and IL-6 (p < 0.001). Silencing the p65 component of NF-κB in A549 cells suppressed the stimulatory effects of influenza virus on secretion of pro-inflammatory cytokines and chemokine. The findings in this study will broaden our understanding of host innate immune mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.

Bone Marrow Cells Expressing Clara Cell Secretory Protein Increase Epithelial Repair After Ablation of Pulmonary Clara Cells

PubMed Central

Bustos, Martha L; Mura, Marco; Marcus, Paula; Hwang, David; Ludkovski, Olga; Wong, Amy P; Waddell, Thomas K

2013-01-01

We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP+ cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP+ cells is beneficial after ablation of lung CCSP+ cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP+ or CCSP− BMC. Compared with mice administered CCSP− cells, mice treated with CCSP+ cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP+ cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP+ BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP+ BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised. PMID:23609017

SGK is a primary glucocorticoid-induced gene in the human.

PubMed

Náray-Fejes-Tóth, A; Fejes-Tóth, G; Volk, K A; Stokes, J B

2000-12-01

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

The role of hERG1 ion channels in epithelial-mesenchymal transition and the capacity of riluzole to reduce cisplatin resistance in colorectal cancer cells.

PubMed

Fortunato, Angelo

2017-08-01

The transition of cells from the epithelial to the mesenchymal state (EMT) plays an important role in tumor progression. EMT allows cells to acquire mobility, stem-like behavior and resistance to apoptosis and drug treatment. These features turn EMT into a central process in tumor biology. Ion channels are attractive targets for the treatment of cancer since they play critical roles in controlling a wide range of physiological processes that are frequently deregulated in cancer. Here, we investigated the role of ether-a-go-go-related 1 (hERG1) ion channels in the EMT of colorectal cancer cells. We studied the epithelial-mesenchymal profile of different colorectal cancer-derived cell lines and the expression of hERG1 potassium channels in these cell lines using real-time PCR. Next, we knocked down hERG1 expression in HCT116 cells using lentivirus mediated RNA interference and characterized the hERG1 silenced cells in vitro and in vivo. Finally, we investigated the capacity of riluzole, an ion channel-modulating drug used in humans to treat amyotrophic lateral sclerosis, to reduce the resistance of the respective colorectal cancer cells to the chemotherapeutic drug cisplatin. We found that of the colorectal cancer-derived cell lines tested, HCT116 showed the highest mesenchymal profile and a high hERG1 expression. Subsequent hERG1 expression knockdown induced a change in cell morphology, which was accompanied by a reduction in the proliferative and tumorigenic capacities of the cells. Notably, we found that hERG1expression knockdown elicited a reversion of the EMT profile in HCT116 cells with a reacquisition of the epithelial-like profile. We also found that riluzole increased the sensitivity of HCT116 cisplatin-resistant cells to cisplatin. Our data indicate that hERG1 plays a role in the EMT of colorectal cancer cells and that its knockdown reduces the proliferative and tumorigenic capacities of these cells. In addition, we conclude that riluzole may be used in combination with cisplatin to reduce chemo-resistance in colorectal cancer cells.

The neoplastic potential of rat tracheal epithelial cell lines induced by dibenzo(a, i)pyrene and 1-nitropyrene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, M.; Ong, T.; Nath, J.

1997-10-01

The rat tracheal epithelial (RTE) cell transformation system is an important short-term assay for respiratory carcinogenesis. In our laboratories, studies have been performed using this assay system to determine the carcinogenic potential of dibenzo(a,i)pyrene (DBP) and 1-nitropyrene (1-NP), two compounds commonly contaminating occupational and environmental settings. RTE cells were exposed in vivo to DBP or 1-NP by intertracheal instillation. RTE cells were then isolated and plated on a medium for determination of cloning and transformation frequencies. Cell lines established from transformed cells induced by DBP and 1-NP were analyzed for their neoplastic potential with the soft agar cloning and themore » athymic nude mouse tumorigenicity assays. Results showed that: (1) incidence of transformed foci in cultures treated with DBP or 1-NP in vivo was significantly higher than that in the control cultures; (2) 8 and 25 cell lines were established from 28 and 48 transformed foci induced by DBP and 1-NP, respectively; (3) 3 of 5 cell lines from DBP and 5 anchorage independent growth in soft agar; (4) some of the cell lines from DBP and 1-NP induced transformed foci formed tumors after cells were injected in athymic nude mice. These results indicate that in vivo exposure to DBP and 1-NP can induce RTE cell transformation and that transformed cells induced by DBP and 1-NP may have neoplastic potential.« less

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

PubMed Central

Band, V; Dalal, S; Delmolino, L; Androphy, E J

1993-01-01

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo. Images PMID:8387914

Conditional Deletion of Pten Causes Bronchiolar Hyperplasia

PubMed Central

Davé, Vrushank; Wert, Susan E.; Tanner, Tiffany; Thitoff, Angela R.; Loudy, Dave E.; Whitsett, Jeffrey A.

2008-01-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (PtenΔ/Δ) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by β-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, β-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles. PMID:17921358

Conditional deletion of Pten causes bronchiolar hyperplasia.

PubMed

Davé, Vrushank; Wert, Susan E; Tanner, Tiffany; Thitoff, Angela R; Loudy, Dave E; Whitsett, Jeffrey A

2008-03-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (Pten(Delta/Delta)) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by beta-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, beta-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles.

NFATC3-PLA2G15 Fusion Transcript Identified by RNA Sequencing Promotes Tumor Invasion and Proliferation in Colorectal Cancer Cell Lines.

PubMed

Jang, Jee-Eun; Kim, Hwang-Phill; Han, Sae-Won; Jang, Hoon; Lee, Si-Hyun; Song, Sang-Hyun; Bang, Duhee; Kim, Tae-You

2018-06-14

This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer lines. We performed paired-end RNA sequencing of 28 colorectal cancer (CRC) cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. 1,380 FT candidates were detected through bioinformatics filtering. We selected 6 candidate FTs, including 4 inter-chromosomal and 2 intra-chromosomal FTs and each FT was found in at least 1 of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in 2. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

PubMed Central

Housley, R M; Morris, C F; Boyle, W; Ring, B; Biltz, R; Tarpley, J E; Aukerman, S L; Devine, P L; Whitehead, R H; Pierce, G F

1994-01-01

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut. Images PMID:7962522

Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma.

PubMed

Shahrabi-Farahani, Shokoufeh; Gallottini, Marina; Martins, Fabiana; Li, Erik; Mudge, Dayna R; Nakayama, Hironao; Hida, Kyoko; Panigrahy, Dipak; D'Amore, Patricia A; Bielenberg, Diane R

2016-04-01

Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Gross, histological and ultrastructural morphology of the aglomerular kidney in the lined seahorse Hippocampus erectus.

PubMed

Fogelson, S B; Yanong, R P E; Kane, A; Teal, C N; Berzins, I K; Smith, S A; Brown, C; Camus, A

2015-09-01

Histologic evaluation of the renal system in the lined seahorse Hippocampus erectus reveals a cranial kidney with low to moderate cellularity, composed of a central dorsal aorta, endothelial lined capillary sinusoids, haematopoietic tissue, fine fibrovascular stroma, ganglia and no nephrons. In comparison, the caudal kidney is moderately to highly cellular with numerous highly convoluted epithelial lined tubules separated by interlacing haematopoietic tissue, no glomeruli, fine fibrovascular stroma, numerous capillary sinusoids, corpuscles of Stannius and clusters of endocrine cells adjacent to large calibre vessels. Ultrastructural evaluation of the renal tubules reveals minimal variability of the tubule epithelium throughout the length of the nephron and the majority of tubules are characterized by epithelial cells with few apical microvilli, elaborate basal membrane infolding, rare electron dense granules and abundant supporting collagenous matrix. © 2015 The Fisheries Society of the British Isles.

Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells

PubMed Central

Huff, Ryan D.; Hsu, Alan C-Y.; Nichol, Kristy S.; Jones, Bernadette; Knight, Darryl A.; Wark, Peter A. B.; Hansbro, Philip M.

2017-01-01

Introduction The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Materials and methods Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. Results HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Conclusions Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines. PMID:28863172

Lipid deposits and lipo-mucosomes in human cholecystitis and epithelial metaplasia in chronic cholecystitis.

PubMed

Gilloteaux, Jacques; Tomasello, Lisa M; Elgison, Deborah A

2003-01-01

Among the inflammatory changes seen in cholecystitis, the ultrastructural alterations of the human gallbladder epithelium include lipid and lipofuscin deposits, fusions of lipid deposits and mucus-containing vesicles forming complex substructural formations called lipo-mucosomes, and microvillar changes of sparse microvilli and basal bodies. Small, lipid-laden structures, such as VLDL-like vesicles, also are fused with the mucus vesicles. Epithelial cell sloughing could liberate and add lipo-mucosomes to the biliary sludge and participate in gallstone formation. With chronic cholelithiasis, fatty degeneration of scattered epithelial cells appears to alter the epithelial lining and favors metaplastic change that could lead to other pathologic changes, including carcinoma in situ-like lesions. In addition to lipid deposition in macrophages, lipid is also incorporated in other cells and tissues of the gallbladder wall (endothelium of capillaries, smooth muscles and fibrocytes).

Restoring conjunctival tolerance by topical nuclear factor-κB inhibitors reduces preservative-facilitated allergic conjunctivitis in mice.

PubMed

Guzmán, Mauricio; Sabbione, Florencia; Gabelloni, María Laura; Vanzulli, Silvia; Trevani, Analía Silvina; Giordano, Mirta Nilda; Galletti, Jeremías Gastón

2014-09-04

To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gopalan, Vinod; Smith, Robert A.; Lam, Alfred K.-Y., E-mail: a.lam@griffith.edu.au

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expressionmore » by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA's effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas. - Highlights: • miR-498 is a non-coding RNA located in 19q13.41. • Colon cancer cell lines showed reduced expression of miR-498. • Mimic driven over expression of miR-498 in colon cancer cells resulted in lower cell proliferation. • miR-498 expression was down regulated in all colorectal adenocarcinoma tissues.« less

Increased ICAM-1 Expression in Transformed Human Oral Epithelial Cells: Molecular Mechanism and Functional Role in Peripheral Blood Mononuclear Cell Adhesion and Lymphokine-Activated-Killer Cell Cytotoxicity

PubMed Central

Huang, George T.-J.; Zhang, Xinli; Park, No-Hee

2012-01-01

The intercellular adhesion molecule-1 (ICAM-1, CD54) serves as a counter-receptor for the β2-integrins, LFA-1 and Mac-1, which are expressed on leukocytes. Although expression of ICAM-1 on tumor cells has a role in tumor progression and development, information on ICAM-1 expression and its role in oral cancer has not been established. Normal human oral keratinocytes (NHOK), human papilloma virus (HPV)-immortalized human oral keratinocyte lines (HOK-16B, HOK-18A, and HOK-18C), and six human oral neoplastic cell lines (HOK-16B-BaP-T1, SCC-4, SCC-9, HEp-2, Tu-177 and 1483) were used to study ICAM-1 expression and its functional role in vitro. Our results demonstrated that NHOK express negligible levels of ICAM-1, whereas immortalized human oral keratinocytes and cancer cells express significantly higher levels of ICAM-1, except for HOK-16B-BaP-T1 and HEp-2. Altered mRNA half-lives did not fully account for the increased accumulation of ICAM-1 mRNA. Adhesion of peripheral blood mononuclear cells (PBMC) to epithelial cells correlated with cell surface ICAM-1 expression levels. This adhesion was inhibited by antibodies specific for either ICAM-1 or LFA-1/Mac-1, suggesting a role for these molecules in adhesion. In contrast, lymphokine-activated-killer (LAK) cell cytotoxic killing of epithelial cells did not correlate with ICAM-1 levels or with adhesion. Nonetheless, within each cell line, blocking of ICAM-1 or LFA-1/Mac-1 reduced LAK cells killing, suggesting that ICAM-1 is involved in mediating this killing. PMID:10938387

Lung Cancer Cell Lines as Tools for Biomedical Discovery and Research

PubMed Central

Girard, Luc; Lockwood, William W.; Lam, Wan L.; Minna, John D.

2010-01-01

Lung cancer cell lines have made a substantial contribution to lung cancer translational research and biomedical discovery. A systematic approach to initiating and characterizing cell lines from small cell and non–small cell lung carcinomas has led to the current collection of more than 200 lung cancer cell lines, a number that exceeds those for other common epithelial cancers combined. The ready availability and widespread dissemination of the lines to investigators worldwide have resulted in more than 9000 citations, including multiple examples of important biomedical discoveries. The high (but not perfect) genomic similarities between lung cancer cell lines and the lung tumor type from which they were derived provide evidence of the relevance of their use. However, major problems including misidentification or cell line contamination remain. Ongoing studies and new approaches are expected to reveal the full potential of the lung cancer cell line panel. PMID:20679594

Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and Epithelial Factors.

PubMed

Tobar, Nicolas; Porras, Omar; Smith, Patricio C; Barros, L Felipe; Martínez, Jorge

2017-01-01

Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long-term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre-culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co-culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136-144, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

NASA Astrophysics Data System (ADS)

Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

2014-04-01

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53-/- mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53-/- mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli.

PubMed

Munne, Pauliina M; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G

2014-04-11

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53(-/-) mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53(-/-) mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

Malignant Mesothelioma—Health Professional Version

Cancer.gov

Epithelial mesothelioma is the most common type of malignant mesothelioma, which forms in the cells that line organs. The other types begin in spindle-shaped cells called sarcomatoid cells or are a mixture of both cell types. Find evidence-based information on malignant mesothelioma treatment.

Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

PubMed Central

Kregel, Steven; Kiriluk, Kyle J.; Rosen, Alex M.; Cai, Yi; Reyes, Edwin E.; Otto, Kristen B.; Tom, Westin; Paner, Gladell P.; Szmulewitz, Russell Z.; Vander Griend, Donald J.

2013-01-01

Despite advances in detection and therapy, castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways, and their regulation by the Androgen Receptor (AR), has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced, castrate-resistant prostate cancers. To this end, we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate, Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative, with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1, endogenous expression of Sox2 was repressed by AR signaling, and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise, in normal prostate epithelial cells and human embryonic stem cells, increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines, and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration, and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways. PMID:23326489

Overexpression of the Ubiquilin-4 (UBQLN4) is Associated with Cell Cycle Arrest and Apoptosis in Human Normal Gastric Epithelial Cell Lines GES-1 Cells by Activation of the ERK Signaling Pathway

PubMed Central

Huang, Shengkai; Dong, Xin; Wang, Jia; Ding, Jie; Li, Yan; Li, Dongdong; Lin, Hong; Wang, Wenjie; Zhao, Mei

2018-01-01

Background Ubiquilin-4 (UBQLN4) is a component of the ubiquitin-proteasome system and regulates the degradation of many proteins implicated in pathological conditions. The aim of this study was to determine the role of UBQLN4 in regulating the proliferation and survival of the normal gastric epithelial cell line GES-1. Material/Methods We constructed GES-1 lines stably overexpressing UBQLN4 by lentiviral infection. Cell proliferation, apoptosis, and the cell cycle were analyzed using the MTT assay and flow cytometric assays. Phosphorylation of ERK, JNK, p38, and expression of cyclin D1 were detected by western blot analysis. Results Overexpression of UBQLN4 significantly reduced proliferation and induced G2/M phase arrest and apoptosis in GES-1 cells. Moreover, upregulation of UBQLN4 increased the expression of cyclin D1 and phosphorylated ERK, but not JNK or p38. Conclusions These data suggest that UBQLN4 may induce cell cycle arrest and apoptosis via activation of the ERK pathway and upregulation of cyclin D1 in GES-1 cells. PMID:29807370

deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium

PubMed Central

Arason, Ari Jon; Jonsdottir, Hulda R.; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K.

2014-01-01

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium. PMID:24533135

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

PubMed

Arason, Ari Jon; Jonsdottir, Hulda R; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K

2014-01-01

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

Primary and Immortalized Human Respiratory Cells Display Different Patterns of Cytotoxicity and Cytokine Release upon Exposure to Deoxynivalenol, Nivalenol and Fusarenon-X

PubMed Central

Ferreira Lopes, Silvia; Vacher, Gaëlle; Savova-Bianchi, Dessislava

2017-01-01

The type B trichothecene mycotoxins deoxynivalenol (DON), nivalenol (NIV) and fusarenon-X (FX) are structurally related secondary metabolites frequently produced by Fusarium on wheat. Consequently, DON, NIV and FX contaminate wheat dusts, exposing grain workers to toxins by inhalation. Those trichothecenes at low, relevant, exposition concentrations have differential effects on intestinal cells, but whether such differences exist with respiratory cells is mostly unknown, while it is required to assess the combined risk of exposure to mycotoxins. The goal of the present study was to compare the effects of DON, NIV and FX alone or in combination on the viability and IL-6 and IL-8-inducing capacity of human epithelial cells representative of the respiratory tract: primary human airway epithelial cells of nasal (hAECN) and bronchial (hAECB) origin, and immortalized human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines. We report that A549 cells are particularly resistant to the cytotoxic effects of mycotoxins. FX is more toxic than DON and NIV for all epithelial cell types. Nasal and bronchial primary cells are more sensitive than bronchial and alveolar cell lines to combined mycotoxin mixtures at low concentrations, although they are less sensitive to mycotoxins alone. Interactions between mycotoxins at low concentrations are rarely additive and are observed only for DON/NIV and NIV/FX on hAECB cells and DON/NIV/FX on A549 cells. Most interactions at low mycotoxin concentrations are synergistic, antagonistic interactions being observed only for DON/FX on hAECB, DON/NIV on 16HBE14o- and NIV/FX on A549 cells. DON, NIV and FX induce, albeit at different levels, IL-6 and IL-8 release by all cell types. However, NIV and FX at concentrations of low cytotoxicity induce IL-6 release by hAECB and A549 cells, and IL-8 release by hAECN cells. Overall, these data suggest that combined exposure to mycotoxins at low concentrations have a stronger effect on primary nasal epithelial cells than on bronchial epithelial cells and activate different inflammatory pathways. This information is particularly relevant for future studies about the hazard of occupational exposure to mycotoxins by inhalation and its impact on the respiratory tract. PMID:29068378

Urban particulate matter increases human airway epithelial cell IL-1β secretion following scratch wounding and H1N1 influenza A exposure in vitro.

PubMed

Hirota, Jeremy A; Marchant, David J; Singhera, Gurpreet K; Moheimani, Fatemeh; Dorscheid, Delbert R; Carlsten, Christopher; Sin, Don; Knight, Darryl

2015-01-01

The airway epithelium represents the first line of defense against inhaled environmental insults including air pollution, allergens, and viruses. Epidemiological and experimental evidence has suggested a link between air pollution exposure and the symptoms associated with respiratory viral infections. We hypothesized that multiple insults integrated by the airway epithelium NLRP3 inflammasome would result in augmented IL-1β release and downstream cytokine production following respiratory virus exposure. We performed in vitro experiments with a human airway epithelial cell line (HBEC-6KT) that involved isolated or combination exposure to mechanical wounding, PM10, house dust mite, influenza A virus, and respiratory syncytial virus. We performed confocal microscopy to image the localization of PM10 within HBEC-6KT and ELISAs to measure soluble mediator production. Airway epithelial cells secrete IL-1β in a time-dependent fashion that is associated with internalization of PM10 particles. PM10 exposure primes human airway epithelial cells to subsequent models of cell damage and influenza A virus exposure. Prior PM10 exposure had no effect on IL-1β responses to RSV exposure. Finally we demonstrate that PM10-priming of human airway epithelial cell IL-1β and GM-CSF responses to influenza A exposure are sensitive to NLRP3 inflammasome inhibition. Our results suggest the NLRP3 inflammasome may contribute to exaggerated immune responses to influenza A virus following periods of poor air quality. Intervention strategies targeting the NLRP3 inflammasome in at risk individuals may restrict poor air quality priming of mucosal immune responses that result from subsequent viral exposures.

Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier.

PubMed

Nazli, Aisha; Dizzell, Sara; Zahoor, Muhammad Atif; Ferreira, Victor H; Kafka, Jessica; Woods, Matthew William; Ouellet, Michel; Ashkar, Ali A; Tremblay, Michel J; Bowdish, Dawn Me; Kaushic, Charu

2018-03-19

More than 40% of HIV infections occur via female reproductive tract (FRT) through heterosexual transmission. Epithelial cells that line the female genital mucosa are the first line of defense against HIV-1 and other sexually transmitted pathogens. These sentient cells recognize and respond to external stimuli by induction of a range of carefully balanced innate immune responses. Previously, we have shown that in response to HIV-1 gp120, the genital epithelial cells (GECs) from upper reproductive tract induce an inflammatory response that may facilitate HIV-1 translocation and infection. In this study, we report that the endometrial and endocervical GECs simultaneously induce biologically active interferon-β (IFNβ) antiviral responses following exposure to HIV-1 that act to protect the epithelial tight junction barrier. The innate antiviral response was directly induced by HIV-1 envelope glycoprotein gp120 and addition of gp120 neutralizing antibody inhibited IFNβ production. Interferon-β was induced by gp120 in upper GECs through Toll-like receptor 2 signaling and required presence of heparan sulfate on epithelial cell surface. The induction of IFNβ was dependent upon activation of transcription factor IRF3 (interferon regulatory factor 3). The IFNβ was biologically active, had a protective effect on epithelial tight junction barrier and was able to inhibit HIV-1 infection in TZM-bl indicator cells and HIV-1 replication in T cells. This is the first report that recognition of HIV-1 by upper GECs leads to induction of innate antiviral pathways. This could explain the overall low infectivity of HIV-1 in the FRT and could be exploited for HIV-1 prophylaxis.Cellular and Molecular Immunology advance online publication, 19 March 2018; doi:10.1038/cmi.2017.168.

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

PubMed

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

2015-08-01

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

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells.

PubMed

Pi, Woojin; Ryu, Jae-Sook; Roh, Jaesook

2011-09-01

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.

IK channel activation increases tumor growth and induces differential behavioral responses in two breast epithelial cell lines.

PubMed

Thurber, Amy E; Nelson, Michaela; Frost, Crystal L; Levin, Michael; Brackenbury, William J; Kaplan, David L

2017-06-27

Many potassium channel families are over-expressed in cancer, but their mechanistic role in disease progression is poorly understood. Potassium channels modulate membrane potential (Vmem) and thereby influence calcium ion dynamics and other voltage-sensitive signaling mechanisms, potentially acting as transcriptional regulators. This study investigated the differential response to over-expression and activation of a cancer-associated potassium channel, the intermediate conductance calcium-activated potassium channel (IK), on aggressive behaviors in mammary epithelial and breast cancer cell lines. IK was over-expressed in the highly metastatic breast cancer cell line MDA-MB-231 and the spontaneously immortalized breast epithelial cell line MCF-10A, and the effect on cancer-associated behaviors was assessed. IK over-expression increased primary tumor growth and metastasis of MDA-MB-231 in orthotopic xenografts, demonstrating for the first time in any cancer type that increased IK is sufficient to promote cancer aggression. The primary tumors had similar vascularization as determined by CD31 staining and similar histological characteristics. Interestingly, despite the increased in vivo growth and metastasis, neither IK over-expression nor activation with agonist had a significant effect on MDA-MB-231 proliferation, invasion, or migration in vitro. In contrast, IK decreased MCF-10A proliferation and invasion through Matrigel but had no effect on migration in a scratch-wound assay. We conclude that IK activity is sufficient to promote cell aggression in vivo. Our data provide novel evidence supporting IK and downstream signaling networks as potential targets for cancer therapies.

Mesenchymal change and drug resistance in neuroblastoma.

PubMed

Naiditch, Jessica A; Jie, Chunfa; Lautz, Timothy B; Yu, Songtao; Clark, Sandra; Voronov, Dimitry; Chu, Fei; Madonna, Mary Beth

2015-01-01

Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells. Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P < 0.1 were analyzed. Immunofluorescence (IF) and Western blot analysis confirmed microarray results of interest. Matrigel invasion assay and migration wounding assays were performed. Volcano plots and heat maps visually demonstrated a similar pattern of DEGs in the SK-N-SH and SK-N-BE(2)C DoxR cell lines relative to their parental WT lines. Venn diagramming revealed 1594 DEGs common to both DoxR cell lines relative to their parental cell lines. Network analysis pointed to several significantly upregulated epithelial-to-mesenchymal transition pathways, through TGF-beta pathways via RhoA, PI3K, and ILK and via SMADs, as well as via notch signaling pathways. DoxR cell lines displayed a more invasive phenotype than respective WT cell lines. Human SK-N-SH and SK-N-BE(2)C NB cells display characteristics of mesenchymal change via multiple pathways in the transition to a drug-resistant state. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2018-01-01

Epithelial surfaces line the body and provide a critical interface between the body and the external environment which is essential to maintaining the symbiotic relationship between the host and the microbiome. Tissue-resident epithelial γδ T cells represent a major T cell population in epithelia and are ideally positioned to perform barrier surveillance and aid in tissue homeostasis and repair. In this review we focus on the intraepithelial γδ compartment in the two largest epithelial tissues in the body, namely the epidermis and intestine, and provide a comprehensive overview of the crucial contributions of intraepithelial γδ cells at these sites to tissue integrity and repair, host homeostasis and host protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we address epithelia-specific butyrophilin-like molecules and touch upon their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires. PMID:28920588

Secretion of Sparfloxacin from the Human Intestinal Caco-2 Cell Line Is Altered by P-Glycoprotein Inhibitors

PubMed Central

Cormet-Boyaka, Estelle; Huneau, Jean-François; Mordrelle, Agnès; Boyaka, Prosper N.; Carbon, Claude; Rubinstein, Ethan; Tomé, Daniel

1998-01-01

The mechanism of intestinal secretion of the difluorinated quinolone sparfloxacin was investigated with the epithelial cell line Caco-2 and was compared to that of the P-glycoprotein (P-gp) substrate vinblastine. The P-gp inhibitors verapamil and progesterone significantly increased the epithelial cell accumulation of both vinblastine and sparfloxacin. This increase is likely to result from an inhibition of drug secretion since both vinblastine uptake and sparfloxacin uptake are known to proceed through a passive transmembrane diffusion. The unidirectional fluxes across cell monlayers grown on permeable filters indicated that a net secretion of sparfloxacin and vinblastine occurred across Caco-2 cells. These secretions were significantly inhibited by the MDR-reversing agent verapamil. We conclude that the P-gp is likely to be involved in the intestinal elimination of the difluorinated quinolone sparfloxacin. PMID:9756763

Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis.

PubMed

Kiuchi, Shizuka; Ikeshita, Shunji; Miyatake, Yukiko; Kasahara, Masanori

2015-02-01

Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity. Copyright © 2014 Elsevier Inc. All rights reserved.

Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation.

PubMed

Nicolson, Garth L; Nawa, Akihiro; Toh, Yasushi; Taniguchi, Shigeki; Nishimori, Katsuhiko; Moustafa, Amr

2003-01-01

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines, we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat mta1 gene, we cloned the human MTA1 gene and found it to be over-expressed in a variety of human cell lines (breast, ovarian, lung, gastric and colorectal cancer but not melanoma or sarcoma) and cancerous tissues (breast, esophageal, colorectal, gastric and pancreatic cancer). We found a close similarity between the human MTA1 and rat mta1 genes (88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively). Both genes encode novel proteins that contain a proline rich region (SH3-binding motif), a putative zinc finger motif, a leucine zipper motif and 5 copies of the SPXX motif found in gene regulatory proteins. Using Southern blot analysis the MTA1 gene was highly conserved, and using Northern blot analysis MTA1 transcripts were found in virtually all human cell lines (melanoma, breast, cervix and ovarian carcinoma cells and normal breast epithelial cells). However, the expression level of the MTA1 gene in normal breast epithelial cells was approximately 50% of that found in rapidly growing adenocarcinoma and atypical epithelial cell lines. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in inhibition of growth and invasion of human MDA-MB-231 breast cancer cells with relatively high MTA1 expression. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected with a mammalian expression vector containing a full-length MTA1 gene. Although some MTA1 protein was found in the cytoplasm, the vast majority of MTA1 protein was localized in the nucleus. Examination of recombinate MTA1 and related MTA2 proteins suggests that MTA1 protein is a histone deacetylase. It also appears to behave like a GATA-element transcription factor, since transfection of a GATA-element reporter into MTA1-expressing cells resulted in 10-20-fold increase in reporter expression over poorly MTA1-expressing cells. Since it was reported that nucleosome remodeling histone deacetylase complex (NuRD complex) involved in chromatin remodeling contains MTA1 protein and a MTA1-related protein (MTA2), we examined NuRD complexes for the presence of MTA1 protein and found an association of this protein with histone deacetylase. The results suggest that the MTA1 protein may serve multiple functions in cellular signaling, chromosome remodeling and transcription processes that are important in the progression, invasion and growth of metastatic epithelial cells.

Robust G2 pausing of adult stem cells in Hydra.

PubMed

Buzgariu, Wanda; Crescenzi, Marco; Galliot, Brigitte

2014-01-01

Hydra is a freshwater hydrozoan polyp that constantly renews its two tissue layers thanks to three distinct stem cell populations that cannot replace each other, epithelial ectodermal, epithelial endodermal, and multipotent interstitial. These adult stem cells, located in the central body column, exhibit different cycling paces, slow for the epithelial, fast for the interstitial. To monitor the changes in cell cycling in Hydra, we established a fast and efficient flow cytometry procedure, which we validated by confirming previous findings, as the Nocodazole-induced reversible arrest of cell cycling in G2/M, and the mitogenic signal provided by feeding. Then to dissect the cycling and differentiation behaviors of the interstitial stem cells, we used the AEP_cnnos1 and AEP_Icy1 transgenic lines that constitutively express GFP in this lineage. For the epithelial lineages we used the sf-1 strain that rapidly eliminates the fast cycling cells upon heat-shock and progressively becomes epithelial. This study evidences similar cycling patterns for the interstitial and epithelial stem cells, which all alternate between the G2 and S-phases traversing a minimal G1-phase. We also found interstitial progenitors with a shorter G2 that pause in G1/G0. At the animal extremities, most cells no longer cycle, the epithelial cells terminally differentiate in G2 and the interstitial progenitors in G1/G0. At the apical pole ~80% cells are post-mitotic differentiated cells, reflecting the higher density of neurons and nematocytes in this region. We discuss how the robust G2 pausing of stem cells, maintained over weeks of starvation, may contribute to regeneration. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Epithelial cell-intrinsic Notch signaling plays an essential role in the maintenance of gut immune homeostasis.

PubMed

Obata, Yuuki; Takahashi, Daisuke; Ebisawa, Masashi; Kakiguchi, Kisa; Yonemura, Shigenobu; Jinnohara, Toshi; Kanaya, Takashi; Fujimura, Yumiko; Ohmae, Masumi; Hase, Koji; Ohno, Hiroshi

2012-03-01

Intestinal epithelial cells (IECs) have important functions as the first line of defense against diverse microorganisms on the luminal surface. Impaired integrity of IEC has been implicated in increasing the risk for inflammatory disorders in the gut. Notch signaling plays a critical role in the maintenance of epithelial integrity by regulating the balance of secretory and absorptive cell lineages, and also by facilitating epithelial cell proliferation. We show in this article that mice harboring IEC-specific deletion of Rbpj (RBP-J(ΔIEC)), a transcription factor that mediates signaling through Notch receptors, spontaneously develop chronic colitis characterized by the accumulation of Th17 cells in colonic lamina propria. Intestinal bacteria are responsible for the development of colitis, because their depletion with antibiotics prevented the development of colitis in RBP-J(ΔIEC) mice. Furthermore, bacterial translocation was evident in the colonic mucosa of RBP-J(ΔIEC) mice before the onset of colitis, suggesting attenuated epithelial barrier functions in these mice. Indeed, RBP-J(ΔIEC) mice displayed increase in intestinal permeability after rectal administration of FITC-dextran. In addition to the defect in physical barrier, loss of Notch signaling led to arrest of epithelial cell turnover caused by downregulation of Hes1, a transcriptional repressor of p27(Kip1) and p57(Kip2). Thus, epithelial cell-intrinsic Notch signaling ensures integrity and homeostasis of IEC, and this mechanism is required for containment of intestinal inflammation.

The composition of the vaginal microbiome in first trimester pregnant women influences the level of autophagy and stress in vaginal epithelial cells.

PubMed

Nasioudis, Dimitrios; Forney, Larry J; Schneider, G Maria; Gliniewicz, Karol; France, Michael T; Boester, Allison; Sawai, Mio; Scholl, Jessica; Witkin, Steven S

2017-09-01

Epithelial cells lining the vagina are major components of genital tract immunity. The influence of the vaginal microbiome on properties of host epithelial cells is largely unexplored. We evaluated whether differences in the most abundant lactobacilli species or bacterial genera in the vagina of first trimester pregnant women were associated with variations in the extent of stress and autophagy in vaginal epithelial cells. Vaginal swabs from 154 first trimester pregnant women were analyzed for bacterial composition by amplification and sequencing of the V1-V3 region of bacterial 16S rRNA genes. Vaginal epithelial cells were lysed and autophagy quantitated by measurement of p62. Intracellular levels of the inducible 70kDa heat shock protein (hsp70), an indicator of cell stress and an autophagy inhibitor, were determined. When Lactobacillus crispatus was the most abundant member of the vaginal microbiota, epithelial p62 and hsp70 levels were lowest as compared to when other bacterial taxa were most abundant. The highest concentrations of p62 and hsp70 were associated with Streptococcus and Bifidobacterium abundance. The p62 level associated with Gardnerella abundance was lower than that observed when lactobacilli other than L. crispatus were most abundant. In conclusion, in the first trimester of pregnancy the abundance of different bacterial taxa is associated with variations in autophagy and magnitude of the stress response in vaginal epithelial cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of claudin species-specific dynamics in reconstitution and remodeling of the zonula occludens.

PubMed

Yamazaki, Yuji; Tokumasu, Reitaro; Kimura, Hiroshi; Tsukita, Sachiko

2011-05-01

Tight-junction strands, which are organized into the beltlike cell-cell adhesive structure called the zonula occludens (TJ), create the paracellular permselective barrier in epithelial cells. The TJ is constructed on the basis of the zonula adherens (AJ) by polymerized claudins in a process mediated by ZO-1/2, but whether the 24 individual claudin family members play different roles at the TJ is unclear. Here we established a cell system for examining the polymerization of individual claudins in the presence of ZO-1/2 using an epithelial-like cell line, SF7, which lacked endogenous TJs and expressed no claudin but claudin-12 in immunofluorescence and real-time PCR assays. In stable SF7-derived lines, exogenous claudin-7, -14, or -19, but no other claudins, individually reconstituted TJs, each with a distinct TJ-strand pattern, as revealed by freeze-fracture analyses. Fluorescence recovery after photobleaching (FRAP) analyses of the claudin dynamics in these and other epithelial cells suggested that slow FRAP-recovery dynamics of claudins play a critical role in regulating their polymerization around AJs, which are loosely coupled with ZO-1/2, to form TJs. Furthermore, the distinct claudin stabilities in different cell types may help to understand how TJs regulate paracellular permeability by altering the paracellular flux and the paracellular ion permeability.

Summer Research Paper

NASA Technical Reports Server (NTRS)

Patel, Zarana

2011-01-01

Certain populations such as chemotherapy patients and atomic bomb survivors have been exposed to ionizing radiation and experience tissue damage and cancer initiation and progression. One cancer that can be initiated from radiation is esophageal squamous cell carcinoma (ESCC), an epithelial cancer that has a survival rate as low as 20%. Researchers have found that when protein tyrosine kinase receptors (RPTK) activate oncogenes, they can create epithelial tumors and cause deadly cancers like ESCC. The RPTK family has one group, MET, that has only two receptors, MET and RON, present in the human body. MET s ligand is the hepatocyte growth factor (HGF) and RON's ligand is the macrophage-stimulating protein (MSP-1). Both HGF and MSP-1 have been shown to activate their receptors and are implicated in certain processes. Since radiation damages cells throughout the biological system, researchers are investigating whether or not HGF and MSP-1 protects or kills certain normal and cancerous cells by being part of cell recovery processes. One research group recently reviewed that the HGF-MET pathway has an important role in the embryonic development in the liver, migration of myogenic precursor cells, regulation of epithelial morphogenesis and growth, and regeneration and protection in tissues. In addition, since the RON receptor is more commonly expressed in cells of epithelial origin, and when activated is part of epithelial cell matrix invasion, dissociation, and migration processes, scientists conclude that RON might be one of the factors causing epithelial cancer initiation in the biological system. In order to examine HGF and MSP-1 s effect on cancer initiation and progression we used two immortalized esophageal epithelial cell lines. One is a normal human cell line (EPC2-hTERT), while the other had a p53 mutation at the 175th amino acid position (EPC2-hTERT-p53(sup R175H)). For this investigation, we used 0(control), 2, and 4 Gray doses of gamma (Cs137) radiation and selected various concentrations from 0-100 ng/mL of HGF and MSP-1 in our assays. Since the HGF and MSP-1 pathways have proliferative roles in epithelial cells, we conducted the MTT proliferation assay to see if either drug enhances or inhibits cell proliferation over time. Also, a MTT cytotoxicity assay was necessary to observe whether the drugs are protecting the cells from radiation and if a trend is occurring depending upon the amount of dose added. In addition, a wound healing assay was done since both drugs have been to known to promote cell motility. Since cell damage occurs when radiation is added, apoptosis and micronuclei assays are vital to see if HGF and MSP-1 increase or decrease cell death and damage in normal and pre-cancerous cells and by how much based on the radiation dosage. Overall, we used the MTT, wound healing, apoptosis and micronuclei assays to investigate the effects ofHGF and MSP-1 on irradiated esophageal epithelial cells.

Generation of urine-derived induced pluripotent stem cells from a patient with phenylketonuria

PubMed Central

Qi, Zijuan; Cui, Yazhou; Shi, Liang; Luan, Jing; Zhou, Xiaoyan; Han, Jinxiang

2018-01-01

Summary The aim of the study was to establish an induced pluripotent stem cell line from urine-derived cells (UiPSCs) from a patient with phenylketonuria (PKU) in order to provide a useful research tool with which to examine the pathology of this rare genetic metabolic disease. Urine-derived epithelial cells (UCs) from a 15-year-old male patient with PKU were isolated and reprogrammed with integration-free episomal vectors carrying an OCT4, SOX2, KLF4, and miR-302-367 cluster. PKU-UiPSCs were verified as correct using alkaline phosphatase staining. Pluripotency markers were detected with real-time PCR and flow cytometry. Promoter methylation in two pluripotent genes, NANOG and OCT4, was analyzed using bisulphite sequencing. An embryoid body (EB) formation assay was also performed. An induced pluripotent stem cell line (iPSC) was generated from epithelial cells in urine from a patient with PKU. This cell line had increased expression of stem cell biomarkers, it efficiently formed EBs, it stained positive for alkaline phosphatase (ALP), and it had a marked decrease in promoter methylation in the NANOG and OCT4 genes. The PKU-UiPSCs created here had typical characteristics and are suitable for further differentiation.

Characterization of an immortalized human vaginal epithelial cell line.

PubMed

Rajan, N; Pruden, D L; Kaznari, H; Cao, Q; Anderson, B E; Duncan, J L; Schaeffer, A J

2000-02-01

Adherence of type 1 piliated Escherichia coli to vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections (UTIs) in women. Progress in understanding the mechanism of adherence to the vaginal surface could be enhanced by the utilization of well-characterized vaginal epithelial cells. The objective of this study was to immortalize vaginal epithelial cells and study their bacterial adherence properties. Primary vaginal cells were obtained from a normal post-menopausal woman, immortalized by infection with E6/E7 genes from human papillomavirus 16 (HPV 16) and cultured in serum free keratinocyte growth factor medium. Positive immunostaining with a pool of antibodies to cytokeratins 1, 5, 10 and 14 (K1, K5, K10 and K14) and to K13 confirmed the epithelial origin of these cells. The immortalized cells showed binding of type 1 piliated E. coli in a pili specific and mannose sensitive manner. This model system should facilitate studies on the interaction of pathogens with vaginal mucosal cells, an essential step in the progression of ascending UTIs in women.

Diacetyl Induces Amphiregulin Shedding in Pulmonary Epithelial Cells and in Experimental Bronchiolitis Obliterans

PubMed Central

Sun, Jesse; Fischer, Bernard M.; Voynow, Judith A.; Kummarapurugu, Apparao B.; Zhang, Helen L.; Nugent, Julia L.; Beasley, Robert F.; Martinu, Tereza; Gwinn, William M.; Morgan, Daniel L.; Palmer, Scott M.

2014-01-01

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air–liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α–converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO. PMID:24816162

Stability of Reference Gene Expression After Porcine Sapelovirus Infection in Porcine Intestinal Epithelial Cells.

PubMed

Huang, Yong; Chen, Yabing; Sun, Huan; Lan, Daoliang

2016-01-01

Intestinal epithelial cells, which serve as the first physical barrier to protect intestinal tract from external antigens, have an important role in the local innate immunity. Screening of reference genes that have stable expression levels after viral infection in porcine intestinal epithelial cells is critical for ensuring the reliability of the expression analysis on anti-infection genes in porcine intestinal epithelial cells. In this study, nine common reference genes in pigs, including ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, TBP, YWHAZ, and RPL32, were chosen as the candidate reference genes. Porcine sapelovirus (PSV) was used as a model virus to infect porcine intestinal epithelial cell line (IPEC-J2). The expression stability of the nine genes was assessed by the geNorm, NormFinder, and BestKeeper software. Moreover, RefFinder program was used to evaluate the analytical results of above three softwares, and a relative expression experiment of selected target gene was used to verify the analysis results. The comprehensive results indicated that the gene combination of TBP and RPL32 has the most stable expression, which could be considered as an appropriate reference gene for research on gene expression after PSV infection in IPEC-J2cells. The results provided essential data for expression analysis of anti-infection genes in porcine intestinal epithelial cells.

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

PubMed

Butler, W B

1984-08-15

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

[Characterization of a human cell line from an anaplastic carcinoma of the thyroid gland].

PubMed

Gioanni, J; Zanghellini, E; Mazeau, C; Zhang, D; Courdi, A; Farges, M; Lambert, J C; Duplay, H; Schneider, M

1991-11-01

A new cell line derived from a thyroid anaplastic carcinoma, CAL 62, has been established in culture. This line is constituted by highly tumorigenic cells. Their epithelial phenotype is stable in culture. Immunochemical staining for human thyroglobulin is negative. Cytogenetic analysis showed a gain of chromosome 20, the translocation i (14q), and breakpoints of centrometric chromatine. These results are similar to those previously reported by other investigators. CAL 62 radiosensibility has been studied. The survival curve of the in vitro irradiated cells has been adjusted with a linear-quadratic model. This cell line is thus showed to be radioresistant. Cell line CAL 62 constitutes an appropriate model for in vitro studies of thyroid anaplastic carcinoma.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

PubMed

Aberdam, Edith; Petit, Isabelle; Sangari, Linda; Aberdam, Daniel

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing

PubMed Central

Aberdam, Edith; Petit, Isabelle; Sangari, Linda

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests. PMID:28640863

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma.

PubMed

Campo-Verde-Arbocco, Fiorella; López-Laur, José D; Romeo, Leonardo R; Giorlando, Noelia; Bruna, Flavia A; Contador, David E; López-Fontana, Gastón; Santiano, Flavia E; Sasso, Corina V; Zyla, Leila E; López-Fontana, Constanza M; Calvo, Juan C; Carón, Rubén W; Creydt, Virginia Pistone

2017-11-07

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.

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

PubMed

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

2013-02-01

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

E-cadherin: A determinant molecule associated with ovarian cancer progression, dissemination and aggressiveness

PubMed Central

Devis, Laura; Lapyckyj, Lara; Besso, María José; Llauradó, Marta; Abascal, María Florencia; Matos, María Laura; Lanau, Lucia; Castellví, Josep; Sánchez, José Luis; Pérez Benavente, Asunción; Gil-Moreno, Antonio; Reventós, Jaume; Santamaria Margalef, Anna; Rigau, Marina; Vazquez-Levin, Mónica Hebe

2017-01-01

Ovarian cancer (OC) is the fifth cancer death cause in women worldwide. The malignant nature of this disease stems from its unique dissemination pattern. Epithelial-to-mesenchymal transition (EMT) has been reported in OC and downregulation of Epithelial cadherin (E-cadherin) is a hallmark of this process. However, findings on the relationship between E-cadherin levels and OC progression, dissemination and aggressiveness are controversial. In this study, the evaluation of E-cadherin expression in an OC tissue microarray revealed its prognostic value to discriminate between advanced- and early-stage tumors, as well as serous tumors from other histologies. Moreover, E-cadherin, Neural cadherin (N-cadherin), cytokeratins and vimentin expression was assessed in TOV-112, SKOV-3, OAW-42 and OV-90 OC cell lines grown in monolayers and under anchorage-independent conditions to mimic ovarian tumor cell dissemination, and results were associated with cell aggressiveness. According to these EMT-related markers, cell lines were classified as mesenchymal (M; TOV-112), intermediate mesenchymal (IM; SKOV-3), intermediate epithelial (IE; OAW-42) and epithelial (E; OV-90). M- and IM-cells depicted the highest migration capacity when grown in monolayers, and aggregates derived from M- and IM-cell lines showed lower cell death, higher adhesion to extracellular matrices and higher invasion capacity than E- and IE-aggregates. The analysis of E-cadherin, N-cadherin, cytokeratin 19 and vimentin mRNA levels in 20 advanced-stage high-grade serous human OC ascites showed an IM phenotype in all cases, characterized by higher proportions of N- to E-cadherin and vimentin to cytokeratin 19. In particular, higher E-cadherin mRNA levels were associated with cancer antigen 125 levels more than 500 U/mL and platinum-free intervals less than 6 months. Altogether, E-cadherin expression levels were found relevant for the assessment of OC progression and aggressiveness. PMID:28934230

Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

NASA Technical Reports Server (NTRS)

Durante, M.; Grossi, G. F.; Gialanella, G.; Pugliese, M.; Nappo, M.; Yang, T. C.

1995-01-01

We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS).

Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells.

PubMed

Durante, M; Grossi, G F; Gialanella, G; Pugliese, M; Nappo, M; Yang, T C

1995-08-01

We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetics Home Reference: hereditary folate malabsorption

MedlinePlus

... PCFT is important for normal functioning of intestinal epithelial cells, which are cells that line the walls of the intestine. ... intestinal absorption and transport into systemic compartments and tissues. Expert Rev Mol Med. 2009 Jan 28;11: ...

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland

PubMed Central

2010-01-01

Introduction Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ)/Smad pathway as an example. Methods We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ. Results IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium. PMID:20942910

Long non-coding RNA ZFAS1 interacts with miR-150-5p to regulate Sp1 expression and ovarian cancer cell malignancy.

PubMed

Xia, Bairong; Hou, Yan; Chen, Hong; Yang, Shanshan; Liu, Tianbo; Lin, Mei; Lou, Ge

2017-03-21

We reported that long non-coding RNA ZFAS1 was upregulated in epithelial ovarian cancer tissues, and was negatively correlated to the overall survival rate of patients with epithelial ovarian cancer in this study. While depletion of ZFAS1 inhibited proliferation, migration, and development of chemoresistance, overexpression of ZFAS1 exhibited an even higher proliferation rate, migration activity, and chemoresistance in epithelial ovarian cancer cell lines. We further found miR-150-5p was a potential target of ZFAS1, which was downregulated in epithelial ovarian cancer tissue. MiR-150-5p subsequently inhibited expression of transcription factor Sp1, as evidence by luciferase assays. Inhibition of miR-150-5p rescued the suppressed proliferation and migration induced by depletion of ZFAS1 in epithelial ovarian cancer cells, at least in part. Taken together, our findings revealed a critical role of ZFAS1/miR-150-5p/Sp1 axis in promoting proliferation rate, migration activity, and development of chemoresistance in epithelial ovarian cancer. And ZFAS1/miR-150-5p may serve as novel markers and therapeutic targets of epithelial ovarian cancer.

Exosomes as mediators of platinum resistance in ovarian cancer.

PubMed

Crow, Jennifer; Atay, Safinur; Banskota, Samagya; Artale, Brittany; Schmitt, Sarah; Godwin, Andrew K

2017-02-14

Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells.

Inhibition of Prolyl Hydroxylase Attenuates Fas Ligand-Induced Apoptosis and Lung Injury in Mice.

PubMed

Nagamine, Yusuke; Tojo, Kentaro; Yazawa, Takuya; Takaki, Shunsuke; Baba, Yasuko; Goto, Takahisa; Kurahashi, Kiyoyasu

2016-12-01

Alveolar epithelial injury and increased alveolar permeability are hallmarks of acute respiratory distress syndrome. Apoptosis of lung epithelial cells via the Fas/Fas ligand (FasL) pathway plays a critical role in alveolar epithelial injury. Activation of hypoxia-inducible factor (HIF)-1 by inhibition of prolyl hydroxylase domain proteins (PHDs) is a possible therapeutic approach to attenuate apoptosis and organ injury. Here, we investigated whether treatment with dimethyloxalylglycine (DMOG), an inhibitor of PHDs, could attenuate Fas/FasL-dependent apoptosis in lung epithelial cells and lung injury. DMOG increased HIF-1α protein expression in vitro in MLE-12 cells, a murine alveolar epithelial cell line. Treatment of MLE-12 cells with DMOG significantly suppressed cell surface expression of Fas and attenuated FasL-induced caspase-3 activation and apoptotic cell death. Inhibition of the HIF-1 pathway by echinomycin or small interfering RNA transfection abolished these antiapoptotic effects of DMOG. Moreover, intraperitoneal injection of DMOG in mice increased HIF-1α expression and decreased Fas expression in lung tissues. DMOG treatment significantly attenuated caspase-3 activation, apoptotic cell death in lung tissue, and the increase in alveolar permeability in mice instilled intratracheally with FasL. In addition, inflammatory responses and histopathological changes were also significantly attenuated by DMOG treatment. In conclusion, inhibition of PHDs protects lung epithelial cells from Fas/FasL-dependent apoptosis through HIF-1 activation and attenuates lung injury in mice.

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katano, Takahito; Ootani, Akifumi; Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501

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 withinmore » 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.« less

The role of miR-200a in mammalian epithelial cell transformation

PubMed Central

Becker, Lindsey E.; Takwi, Apana Agha L.; Lu, Zhongxin; Li, Yong

2015-01-01

Cancer is a multistep disease that begins with malignant cell transformation and frequently culminates in metastasis. MicroRNAs (miRNAs) are small regulatory 21–25 nt RNA molecules and are frequently deregulated in cancer. miR-200a is a member of the miR-200 family, which are known inhibitors of the epithelial-to-mesenchymal transition. As such, the tumor-suppressive role of miR-200a in oncogenesis has been well documented; however, recent studies have found a proliferative role for this miRNA as well as a prometastatic role in the later steps of cancer progression. Little is known about the role of this miRNA in the early stages of cancer, namely, malignant cell transformation. Here, we show that miR-200a alone transforms an immortalized rat epithelial cell line, and miR-200a cooperates with Ras to enhance malignant transformation of an immortalized human epithelial cell line. Furthermore, miR-200a induces cell transformation and tumorigenesis in immunocompromised mice by cooperating with a Ras mutant that activates only the RalGEF effector pathway, but not Ras mutants activating PI3K or Raf effector pathways. This transformative ability is in accordance with miR-200a targeting Fog2 and p53 to activate Akt and directly repress p53 protein levels, respectively. These results demonstrate an oncogenic role for miR-200a and provide a specific cellular context where miR-200a acts as an oncomiR rather than a tumor suppressor by cooperating with an oncogene in malignant cell transformation. PMID:25239643

AFRRI (Armed Forces Radiobiology Research Institute) Reports, January- March 1986

DTIC Science & Technology

1986-03-01

cells , calculated from —50 to —40 mV, wap 101 nS4:0-12 (mean+ 8.E. of mean, n = 22 ). 480 E. K. GAL UN AND P. A. SHEEH Y Effect of adherence on zero...and Hagan, M. Effect of radiation on the regulation of sodium-dependent glucose transport in LLC-PKi epithelial cell line: Possible model for...epithelial cells . SR86-9: Mullin, M. J., and Hunt, W. A. Actions of ethanol on voltage-sensitive sodium channels: Effects on neurotoxin-stimulated

Immunoelectron microscopic localisation of keratin and luminal epithelial antigens in normal and neoplastic urothelium.

PubMed

Wilson, P D; Nathrath, W B; Trejdosiewicz, L K

1982-01-01

Immunoelectron microscope cytochemistry was carried out on 2% paraformaldehyde fixed, 50 mu sections of normal urothelium and bladder carcinoma cells in culture using antisera raised in rabbits to human 40-63 000 MW epidermal "broad spectrum" keratin and calf urothelial "luminal epithelial antigen" (aLEA) Both the unconjugated and indirect immunoperoxidase-DAB techniques were used before routine embedding. The localisation of both keratin and luminal epithelial antigen (LEA) was similar in normal and neoplastic cells and reaction product was associated not only with tonofilaments but also lining membrane vesicles and on fine filaments in the cytoplasmic ground substance.

Hydrogen Peroxide Contributes to the Epithelial Cell Death Induced by the Oral Mitis Group of Streptococci

PubMed Central

Okahashi, Nobuo; Sumitomo, Tomoko; Nakata, Masanobu; Sakurai, Atsuo; Kuwata, Hirotaka; Kawabata, Shigetada

2014-01-01

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H2O2) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H2O2 may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H2O2-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited S. oralis cytotoxicity, and H2O2 alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H2O2 production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H2O2 induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H2O2 is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts. PMID:24498253

Expression of Ki-67 in odontogenic cysts: A comparative study between odontogenic keratocysts, radicular cysts and dentigerous cysts.

PubMed

Modi, Tapan G; Chalishazar, Monali; Kumar, Malay

2018-01-01

Odontogenic cysts are the most common cysts of the jaws and are formed from the remnants of the odontogenic apparatus. Among these odontogenic cysts, radicular cysts (RCs) (about 60% of all diagnosed jaw cysts), dentigerous cysts (DCs) (16.6% of all jaw cysts) and odontogenic keratocysts (OKCs) (11.2% of all developmental odontogenic cysts) are the most common. The behavior of any lesion is generally reflected by its growth potential. Growth potential is determined by measuring the cell proliferative activity. The cell proliferative activity is measured by various methods among which immunohistochemistry (IHC) is the commonly used technique. Most of the IHC studies on cell proliferation have been based on antibodies such as Ki-67 and proliferating cell nuclear antigen. In the present study, the total sample size comprised of 45 cases of odontogenic cysts, with 15 cases each of OKC, RC and DC. Here, an attempt is made to study immunohistochemical (streptavidin-biotin detection system HRP-DAB) method to assess the expression of Ki-67 in different layers of the epithelial lining of OKCs, RCs and DCs. Ki-67 positive cells were highest in epithelium of OKC as compared to DC and RC. The increased Ki-67 labeling index and its expression in suprabasal cell layers of epithelial lining in OKC and its correlation with suprabasal cell layers of epithelial lining in DC and RC could contribute toward its clinically aggressive behavior. OKC is of more significance to the oral pathologist and oral surgeon because of its specific histopathological features, high recurrence rate and aggressive behavior.

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri

PubMed Central

MATSUO, Yosuke; MIYOSHI, Yukihiro; OKADA, Sanae; SATOH, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion. PMID:24936355

Transformation of Mouse Liver Cells by Methylcholanthrene Leads to Phenotypic Changes Associated with Epithelial-mesenchymal Transition.

PubMed

Oh, Jiyun; Kwak, Jae-Hwan; Kwon, Do-Young; Kim, A-Young; Oh, Dal-Seok; Je, Nam Kyung; Lee, Jaewon; Jung, Young-Suk

2014-12-01

Environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) have been implicated in cancer development and progression. However, the effects of PAHs on carcinogenesis are still poorly understood. Here, we characterized a mouse cancer cell line BNL 1ME A. 7R.1 (1MEA) derived by transformation of non-tumorigenic liver cell line BNL CL.2 (BNL) using 3-methylcholanthrene (3MC), a carcinogenic PAH. RT-PCR and immunoblot analysis were used to determine the expression level of mRNA and proteins, respectively. To determine functionality, cell motility was assessed in vitro using a transwell migration assay. Both mRNA and protein levels of E-cadherin were significantly decreased in 1MEA cells in comparison with BNL cells. While the expression levels of mesenchymal markers and related transcription factors were enhanced in 1MEA cells, which could lead to increase in cell motility. Indeed, we found that 7-day exposure of BNL cells to 3-MC reduced the level of the adhesion molecule and epithelial marker Ecadherin and increased reciprocally the level of the mesenchymal marker vimentin in a dose-dependent manner. Taken together, these results indicate that the process of epithelial-mesenchymal transition (EMT) may be activated during premalignant transformation induced by 3-MC. A mechanism study to elucidate the relation between 3-MC exposure and EMT is underway in our laboratory.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines.

PubMed

Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E; Krishnan, Aswini R; Tsui, Tzuhan; Aguilera, Joseph A; Advani, Sunil; Crotty Alexander, Laura E; Brumund, Kevin T; Wang-Rodriguez, Jessica; Ongkeko, Weg M

2016-01-01

Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

PubMed Central

Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E.; Krishnan, Aswini R.; Tsui, Tzuhan; Aguilera, Joseph A.; Advani, Sunil; Crotty Alexander, Laura E.; Brumund, Kevin T.; Wang-Rodriguez, Jessica

2016-01-01

Objectives Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. Materials and Methods HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 hours to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. Results E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. Conclusion E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. PMID:26547127

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

PubMed

Ishikawa, F; Ushida, K; Mori, K; Shibanuma, M

2015-01-22

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT ((Tert)HMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached (Tert)HMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.

Fucosylation and protein glycosylation create functional receptors for cholera toxin

PubMed Central

Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E; Cervin, Jakob; Dedic, Benjamin; Rodriguez, Andrea C; Nischan, Nicole; Bond, Michelle R; Mettlen, Marcel; Trudgian, David C; Lemoff, Andrew; Quiding-Järbrink, Marianne; Gustavsson, Bengt; Steentoft, Catharina; Clausen, Henrik; Mirzaei, Hamid; Teneberg, Susann; Yrlid, Ulf; Kohler, Jennifer J

2015-01-01

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera. DOI: http://dx.doi.org/10.7554/eLife.09545.001 PMID:26512888

Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Di Kaijun; Wong, Y.C.; Wang Xianghong

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation ofmore » MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.« less

Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.

PubMed

Kojima, Takashi; Takasawa, Akira; Kyuno, Daisuke; Ito, Tatsuya; Yamaguchi, Hiroshi; Hirata, Koichi; Tsujiwaki, Mitsuhiro; Murata, Masaki; Tanaka, Satoshi; Sawada, Norimasa

2011-10-01

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

S-carboxymethylcysteine inhibits adherence of Streptococcus pneumoniae to human alveolar epithelial cells.

PubMed

Sumitomo, Tomoko; Nakata, Masanobu; Yamaguchi, Masaya; Terao, Yutaka; Kawabata, Shigetada

2012-01-01

Streptococcus pneumoniae is a major pathogen of respiratory infections that utilizes platelet-activating factor receptor (PAFR) for firm adherence to host cells. The mucolytic agent S-carboxymethylcysteine (S-CMC) has been shown to exert inhibitory effects against infection by several respiratory pathogens including S. pneumoniae in vitro and in vivo. Moreover, clinical studies have implicated the benefits of S-CMC in preventing exacerbation of chronic obstructive pulmonary disease, which is considered to be related to respiratory infections. In this study, to assess whether the potency of S-CMC is attributable to inhibition of pneumococcal adherence to host cells, an alveolar epithelial cell line stimulated with interleukin-1α was used as a model of inflamed epithelial cells. Despite upregulation of PAFR by inflammatory activation, treatment with S-CMC efficiently inhibited pneumococcal adherence to host epithelial cells. In order to gain insight into the inhibitory mechanism, the effects of S-CMC on PAFR expression were also investigated. Following treatment with S-CMC, PAFR expression was reduced at both mRNA and post-transcriptional levels. Interestingly, S-CMC was also effective in inhibiting pneumococcal adherence to cells transfected with PAFR small interfering RNAs. These results indicate S-CMC as a probable inhibitor targeting numerous epithelial receptors that interact with S. pneumoniae.

CXCR3 chemokine receptor-induced chemotaxis in human airway epithelial cells: role of p38 MAPK and PI3K signaling pathways.

PubMed

Shahabuddin, Syed; Ji, Rong; Wang, Ping; Brailoiu, Eugene; Dun, Na; Yang, Yi; Aksoy, Mark O; Kelsen, Steven G

2006-07-01

Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 +/- 88% (mean +/- SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca(2+)] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5-10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

PubMed

Joo, Jin Woo; Kim, Hyun-Soo; Do, Sung-Im; Sung, Ji-Youn

2018-05-01

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Isolation and functional studies of human fetal gastric epithelium in primary culture.

PubMed

Chailler, Pierre; Beaulieu, Jean-François; Ménard, Daniel

2012-01-01

Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and devoid of glandular zymogenic chief cells. We successfully applied a new nonenzymatic procedure using Matrisperse Cell Recovery Solution to dissociate the entire epithelium from human fetal stomach. Cultures were generated by seeding multicellular aggregates prepared by mechanical fragmentation. We further demonstrate that this simple and convenient technique allows for the maintenance of heterogenous gastric epithelial primary cultures on plastic without a biological matrix as well as the persistence of viable chief cells able to synthesize and secrete gastric digestive enzymes, i.e., pepsinogen and gastric lipase. In wounding experiments, epithelial restitution occurred in serum-reduced conditions and was modulated by exogenous agents. This culture system is thus representative of the foveolus-gland axis and offers new perspectives to establish the influence of individual growth factors and extracellular matrix components as well as their combinatory effects on gastric epithelium homeostasis.

Downregulation of Bit1 expression promotes growth, anoikis resistance, and transformation of immortalized human bronchial epithelial cells via Erk activation-dependent suppression of E-cadherin.

PubMed

Yao, Xin; Gray, Selena; Pham, Tri; Delgardo, Mychael; Nguyen, An; Do, Stephen; Ireland, Shubha Kale; Chen, Renwei; Abdel-Mageed, Asim B; Biliran, Hector

2018-01-01

The mitochondrial Bit1 protein exerts tumor-suppressive function in NSCLC through induction of anoikis and inhibition of EMT. Having this dual tumor suppressive effect, its downregulation in the established human lung adenocarcinoma A549 cell line resulted in potentiation of tumorigenicity and metastasis in vivo. However, the exact role of Bit1 in regulating malignant growth and transformation of human lung epithelial cells, which are origin of most forms of human lung cancers, has not been examined. To this end, we have downregulated the endogenous Bit1 expression in the immortalized non-tumorigenic human bronchial epithelial BEAS-2B cells. Knockdown of Bit1 enhanced the growth and anoikis insensitivity of BEAS-2B cells. In line with their acquired anoikis resistance, the Bit1 knockdown BEAS-2B cells exhibited enhanced anchorage-independent growth in vitro but failed to form tumors in vivo. The loss of Bit1-induced transformed phenotypes was in part attributable to the repression of E-cadherin expression since forced exogenous E-cadherin expression attenuated the malignant phenotypes of the Bit1 knockdown cells. Importantly, we show that the loss of Bit1 expression in BEAS-2B cells resulted in increased Erk activation, which functions upstream to promote TLE1-mediated transcriptional repression of E-cadherin. These collective findings indicate that loss of Bit1 expression contributes to the acquisition of malignant phenotype of human lung epithelial cells via Erk activation-induced suppression of E-cadherin expression. Copyright © 2017 Elsevier Inc. All rights reserved.

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

NASA Technical Reports Server (NTRS)

Wang, Minli; Hada, Megumi; Huff, Janice; Pluth, Janice M.; Anderson, Janniffer; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.

PubMed

Qian, Jiaying; Niu, Jiangong; Li, Ming; Chiao, Paul J; Tsao, Ming-Sound

2005-06-15

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

Transformation of Epithelial Ovarian Cancer Stemlike Cells into Mesenchymal Lineage via EMT Results in Cellular Heterogeneity and Supports Tumor Engraftment

PubMed Central

Jiang, Hua; Lin, Xiaolong; Liu, Yingtao; Gong, Wenjia; Ma, Xiaoling; Yu, Yinhua; Xie, Yi; Sun, Xiaoxi; Feng, Youji; Janzen, Viktor; Chen, Tong

2012-01-01

Ovarian cancers are heterogeneous and contain stemlike cells that are able to self-renew and are responsible for sustained tumor growth. Metastasis in the peritoneal cavity occurs more frequently in ovarian cancer than in other malignancies, but the underlying mechanism remains largely unknown. We have identified that ovarian cancer stemlike cells (CSCs), which were defined as side population (SP) cells, were present in patients’ ascitic fluid and mesenchymally transformed cell lines, ES-2 and HO-8910PM. SP cells, which were sorted from both cell lines and implanted into immunocompromised mice, were localized to the xenografted tumor boundary. In addition, SP cells exhibited an epithelial phenotype and showed a distinct gene expression profile with reduced expression of cell adhesion molecules (CAMs), indicating that SP cells exert an important role in ovarian cancer progression on the basis of their delicate interaction with the surrounding microenvironment and anatomical localization in tumors. In contrast, non-SP cells exhibited a more mesenchymal phenotype and showed more increased invasive potential than SP cells. This heterogeneity was observed as an endogenous transformation via the epithelial–mesenchymal transition (EMT) process. Inhibition of the EMT process by Snail1 silencing reduced the SP cell frequency, and affected their invasive capacity and engraftment. These findings illustrate the interplay between epithelial ovarian CSCs and the EMT, and exert a link to explain tumor heterogeneity and its necessity for ovarian cancer maintenance, metastasis and progression. PMID:22801793

Mithramycin inhibits epithelial-to-mesenchymal transition and invasion by downregulating SP1 and SNAI1 in salivary adenoid cystic carcinoma.

PubMed

Li, Jiasu; Gao, Hongmei; Meng, Lingxu; Yin, Lin

2017-06-01

Mithramycin exhibits certain anticancer effects in glioma, metastatic cerebral carcinoma, malignant lymphoma, chorionic carcinoma and breast cancer. However, its effects on salivary adenoid cystic carcinoma remain unclear. Here, we report that mithramycin significantly inhibited epithelial-to-mesenchymal transition and invasion in human salivary adenoid cystic carcinoma cell lines. The underlying mechanism for this activity was further demonstrated to involve decreasing the expression of the transcription factors specificity protein 1 and SNAI1. Specificity protein 1 is a pro-tumourigenic transcription factor that is overexpressed in SACC-LM and SACC-83 cells, and its expression is inhibited by mithramycin. Moreover, chromatin immunoprecipitation assays showed that specificity protein 1 induced SNAI1 transcription through direct binding to the SNAI1 promoter. In summary, this study uncovered the mechanism through which mithramycin inhibits epithelial-to-mesenchymal transition and invasion in salivary adenoid cystic carcinoma cell lines, namely, via downregulating specificity protein 1 and SNAI1 expression, which suggests mithramycin may be a promising therapeutic option for salivary adenoid cystic carcinoma.

Targeting Androgen Receptor to Suppress Macrophage-induced EMT and Benign Prostatic Hyperplasia (BPH) Development

PubMed Central

Lu, Tianjing; Lin, Wen-Jye; Izumi, Kouji; Wang, Xiaohai; Xu, Defeng; Fang, Lei-Ya; Li, Lei; Jiang, Qi

2012-01-01

Early studies suggested macrophages might play roles in inflammation-associated benign prostatic hyperplasia (BPH) development, yet the underlying mechanisms remain unclear. Here we first showed that CD68+ macrophages were identified in both epithelium and the stromal area of human BPH tissues. We then established an in vitro co-culture model with prostate epithelial and macrophage cell lines to study the potential impacts of infiltrating macrophages in the BPH development and found that co-culturing prostate epithelial cells with macrophages promoted migration of macrophages. In a three-dimensional culture system, the sphere diameter of BPH-1 prostate cells was significantly increased during coculture with THP-1 macrophage cells. Mechanism dissection suggested that expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, Snail, and TGF-β2, were increased, and administration of anti-TGF-β2 neutralizing antibody during co-culture suppressed the EMT and THP-1-mediated growth of BPH-1 cells, suggesting THP-1 might go through EMT to influence the BPH development and progression. Importantly, we found that modulation of androgen receptor (AR) in BPH-1 and mPrE cells significantly increased THP-1 and RAW264.7 cell migration, respectively, and enhanced expression levels of EMT markers, suggesting that AR in prostate epithelial cells might play a role in promoting macrophage-mediated EMT in prostate epithelial cells. Silencing AR function via an AR degradation enhancer, ASC-J9, decreased the macrophage migration to BPH-1 cells and suppressed EMT marker expression. Together, these results provide the first evidence to demonstrate that prostate epithelial AR function is important for macrophage-mediated EMT and proliferation of prostate epithelial cells, which represents a previously unrecognized role of AR in the cross-talk between macrophages and prostate epithelial cells. These results may provide new insights for a new therapeutic approach to battle BPH via targeting AR and AR-mediated inflammatory signaling pathways. PMID:22915828

Targeting androgen receptor to suppress macrophage-induced EMT and benign prostatic hyperplasia (BPH) development.

PubMed

Lu, Tianjing; Lin, Wen-Jye; Izumi, Kouji; Wang, Xiaohai; Xu, Defeng; Fang, Lei-Ya; Li, Lei; Jiang, Qi; Jin, Jie; Chang, Chawnshang

2012-10-01

Early studies suggested macrophages might play roles in inflammation-associated benign prostatic hyperplasia (BPH) development, yet the underlying mechanisms remain unclear. Here we first showed that CD68(+) macrophages were identified in both epithelium and the stromal area of human BPH tissues. We then established an in vitro co-culture model with prostate epithelial and macrophage cell lines to study the potential impacts of infiltrating macrophages in the BPH development and found that co-culturing prostate epithelial cells with macrophages promoted migration of macrophages. In a three-dimensional culture system, the sphere diameter of BPH-1 prostate cells was significantly increased during coculture with THP-1 macrophage cells. Mechanism dissection suggested that expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, Snail, and TGF-β2, were increased, and administration of anti-TGF-β2 neutralizing antibody during co-culture suppressed the EMT and THP-1-mediated growth of BPH-1 cells, suggesting THP-1 might go through EMT to influence the BPH development and progression. Importantly, we found that modulation of androgen receptor (AR) in BPH-1 and mPrE cells significantly increased THP-1 and RAW264.7 cell migration, respectively, and enhanced expression levels of EMT markers, suggesting that AR in prostate epithelial cells might play a role in promoting macrophage-mediated EMT in prostate epithelial cells. Silencing AR function via an AR degradation enhancer, ASC-J9, decreased the macrophage migration to BPH-1 cells and suppressed EMT marker expression. Together, these results provide the first evidence to demonstrate that prostate epithelial AR function is important for macrophage-mediated EMT and proliferation of prostate epithelial cells, which represents a previously unrecognized role of AR in the cross-talk between macrophages and prostate epithelial cells. These results may provide new insights for a new therapeutic approach to battle BPH via targeting AR and AR-mediated inflammatory signaling pathways.

Intracellular Kinases Mediate Increased Translation and Secretion of Netrin-1 from Renal Tubular Epithelial Cells

PubMed Central

Jayakumar, Calpurnia; Mohamed, Riyaz; Ranganathan, Punithavathi Vilapakkam; Ramesh, Ganesan

2011-01-01

Background Netrin-1 is a laminin-related secreted protein, is highly induced after tissue injury, and may serve as a marker of injury. However, the regulation of netrin-1 production is not unknown. Current study was carried out in mouse and mouse kidney cell line (TKPTS) to determine the signaling pathways that regulate netrin-1 production in response to injury. Methods and Principal Findings Ischemia reperfusion injury of the kidney was induced in mice by clamping renal pedicle for 30 minutes. Cellular stress was induced in mouse proximal tubular epithelial cell line by treating with pervanadate, cisplatin, lipopolysaccharide, glucose or hypoxia followed by reoxygenation. Netrin-1 expression was quantified by real time RT-PCR and protein production was quantified using an ELISA kit. Cellular stress induced a large increase in netrin-1 production without increase in transcription of netrin-1 gene. Mitogen activated protein kinase, ERK mediates the drug induced netrin-1 mRNA translation increase without altering mRNA stability. Conclusion Our results suggest that netrin-1 expression is suppressed at the translational level and MAPK activation leads to rapid translation of netrin-1 mRNA in the kidney tubular epithelial cells. PMID:22046354

Mixed epithelial and stromal tumor of the middle ear: The first case report.

PubMed

Michal, Michael; Skálová, Alena; Kazakov, Dmitry V; Pecková, Květoslava; Heidenreich, Filip; Grossmann, Petr; Michal, Michal

2017-03-01

We report a tumor arising in the middle ear of a 65-year-old female patient that was composed of an ovarian-type stroma (OS) and an epithelial component. The tumor consisted of irregular, polypoid masses containing multiple variably sized cystic spaces, which were invariably surrounded by the OS. The cystic spaces were lined by flat, cuboidal, or columnar epithelial cells, in most parts showing mucinous differentiation. The epithelial lining of the cysts strongly expressed cytokeratins AE1-3, CK7, CK8, CK18, CK19, EMA, and S100 protein. The stroma expressed CD34 and smooth muscle actin. No cytological atypia or mitoses were present, and the proliferative activity was less than 1% in both components. The clonality analysis proved the clonal nature of the neoplasm. We believe that this tumor is a new member in the family of neoplasms containing the OS, and therefore we propose the term mixed epithelial and stromal tumor of the middle ear. Copyright © 2017 Elsevier Inc. All rights reserved.

The relationship between cisplatin resistance and histone deacetylase isoform overexpression in epithelial ovarian cancer cell lines

PubMed Central

Kim, Min-Gyun; Pak, Jhang Ho; Choi, Won Ho; Park, Jeong-Yeol; Nam, Joo-Hyun

2012-01-01

Objective To investigate the relationship between cisplatin resistance and histone deacetylase (HDAC) isoform overexpression in ovarian cancer cell lines. Methods Expression of four HDAC isoforms (HDAC 1, 2, 3, and 4) in two ovarian cancer cell lines, SKOV3 and OVCAR3, exposed to various concentrations of cisplatin was examined by western blot analyses. Cells were transfected with plasmid DNA of each HDAC. The overexpression of protein and mRNA of each HDAC was confirmed by western blot and reverse transcriptase-polymerase chain reaction analyses, respectively. The cell viability of the SKOV3 and OVCAR3 cells transfected with HDAC plasmid DNA was measured using the cell counting kit-8 assay after treatment with cisplatin. Results The 50% inhibitory concentration of the SKOV3 and OVCAR3 cells can be determined 15-24 hours after treatment with 15 µg/mL cisplatin. The expression level of acetylated histone 3 protein in SKOV3 cells increased after exposure to cisplatin. Compared with control cells at 24 hours after cisplatin exposure, the viability of SKOV3 cells overexpressing HDAC 1 and 3 increased by 15% and 13% (p<0.05), respectively. On the other hand, OVCAR3 cells that overexpressed HDAC 2 and 4 exhibited increased cell viability by 23% and 20% (p<0.05), respectively, compared with control cells 24 hours after exposure to cisplatin. Conclusion In SKOV3 and OVCAR3 epithelial ovarian cancer cell lines, the correlation between HDAC overexpression and cisplatin resistance was confirmed. However, the specific HDAC isoform associated with resistance to cisplatin varied depending on the ovarian cancer cell line. These results may suggest that each HDAC isoform conveys cisplatin resistance via different mechanisms. PMID:22808361

Generation of a TALEN-mediated, p63 knock-in in human induced pluripotent stem cells.

PubMed

Kobayashi, Yuki; Hayashi, Ryuhei; Quantock, Andrew J; Nishida, Kohji

2017-12-01

The expression of p63 in surface ectodermal cells during development of the cornea, skin, oral mucosa and olfactory placodes is integral to the process of cellular self-renewal and the maintenance of the epithelial stem cell status. Here, we used TALEN technology to generate a p63 knock-in (KI) human induced pluripotent stem (hiPS) cell line in which p63 expression can be visualized via enhanced green fluorescent protein (EGFP) expression. The KI-hiPS cells maintained pluripotency and expressed the stem cell marker gene, ΔNp63α. They were also able to successfully differentiate into functional corneal epithelial cells as assessed by p63 expression in reconstructed corneal epithelium. This approach enables the tracing of p63-expressing cell lineages throughout epithelial development, and represents a promising application in the field of stem cell research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Ovarian Epithelial Inclusions With Mucinous Differentiation: A Clinicopathologic Study of 42 Cases.

PubMed

Seidman, Jeffrey D; Krishnan, Jayashree

2017-07-01

Ovarian epithelial inclusions lined by mucinous epithelium are rare and of uncertain origin. Ovaries containing such inclusions were studied in 42 women. The inclusions were divided into 3 groups: serous epithelial lined with typical ciliated morphology but with distinct basophilic cytoplasmic mucin in some or all of the lining cells, those lined by typical mucinous epithelium, and those lined by a combination of typical mucinous epithelium and serous epithelium. The mean patient age was 61.5 years. Pure mucinous inclusions were found in 27 patients, serous-type inclusions with cytoplasmic mucin in 20, and mixed type in 10. All 3 types of inclusions were found in 1 patient. Two types of inclusions were found in 13. Four patients had associated mucinous neoplasms (1 mucinous cystadenoma, 1 atypical proliferative seromucinous tumor, and 2 seromucinous cystadenomas), and 11 patients (26%) had endometriosis. The fallopian tubes in 4 patients (9.5%) also displayed mucinous metaplasia; this was not significantly different from the 3.1% we found in our previously reported series of unselected tubes from the same population. These findings suggest that mucinous inclusions may arise as a direct metaplastic change in serous-type inclusions. Other possible origins of mucinous inclusions in the ovarian cortex include endometriosis and Brenner (transitional cell) nests. Whether such inclusions can be a source of mucinous ovarian neoplasms as are Brenner tumors and mature cystic teratomas is unknown and may warrant further investigation.

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp; Naganuma, Kaori; Kato, Kenichi

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histonemore » H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.« less

The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress.

PubMed

Andley, U P; Song, Z; Wawrousek, E F; Bassnett, S

1998-11-20

alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular functions in the lens over and above its well characterized role in refraction.

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin.

PubMed

Sánchez, O; Montesino, R; Toledo, J R; Rodríguez, E; Díaz, D; Royle, L; Rudd, P M; Dwek, R A; Gerwig, G J; Kamerling, J P; Harvey, D J; Cremata, J A

2007-08-15

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.

Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

PubMed

Stein, Daniel C; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

2015-01-01

Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

Bag3 promotes resistance to apoptosis through Bcl-2 family members in non-small cell lung cancer.

PubMed

Zhang, Yong; Wang, Jian-Hua; Lu, Qiang; Wang, Yun-Jie

2012-01-01

In non-small cell lung cancer (NSCLC) certain molecular characteristics, which are related to molecular alterations have been investigated. These are responsible for both the initiation and maintenance of the malignancy in lung cancer. The aim of this study was to evaluate the influence of Bag3 (Bcl-2 associated athanogene 3) in the regulation of apoptosis on NSCLC. Bag3 and Hsp70 expression were examined by immunohistochemistry to confirm their potential roles in the prevalence of NSCLC. We also established human normal bronchial epithelial cells and HOP-62 cell line as the model to analyze cell apoptosis and the expression of Hsp70, Bcl-XL and Bcl-2, which were affected by Bag3. In this study, we found that Bag3 and Hsp70 are highly expressed in few tissues and cell lines of NSCLC. Bag3 inhibits apoptosis in human normal bronchial epithelial cell lines and sustain the survival of NSCLC cells. Bag3, Hsp70, Bcl-XL and Bcl-2 are up-regulated in NSCLC cell lines. At the same time, the silencing of Bag3 results in diminishing protein levels of Bcl-XL and Bcl-2. The results of immunoprecipitation identified that Bag3 could interact with Hsp70, Bcl-XL and Bcl-2 NSCLC cells directly or indirectly. We conclude that NSCLC cells were protected from apoptosis through increasing Bag3 expression and consequently promoted the expression of Bcl-XL and Bcl-2.

Oncogenic ALK regulates EMT in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1.

PubMed

Voena, Claudia; Varesio, Lydia M; Zhang, Liye; Menotti, Matteo; Poggio, Teresa; Panizza, Elena; Wang, Qi; Minero, Valerio G; Fagoonee, Sharmila; Compagno, Mara; Altruda, Fiorella; Monti, Stefano; Chiarle, Roberto

2016-05-31

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.

Oncogenic ALK regulates EMT in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1

PubMed Central

Menotti, Matteo; Poggio, Teresa; Panizza, Elena; Wang, Qi; Minero, Valerio G.; Fagoonee, Sharmila; Compagno, Mara; Altruda, Fiorella; Monti, Stefano; Chiarle, Roberto

2016-01-01

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy. PMID:27119231

Budesonide suppresses pulmonary antibacterial host defense by down-regulating cathelicidin-related antimicrobial peptide in allergic inflammation mice and in lung epithelial cells

PubMed Central

2013-01-01

Background Glucocorticoids are widely regarded as the most effective treatment for asthma. However, the direct impact of glucocorticoids on the innate immune system and antibacterial host defense during asthma remain unclear. Understanding the mechanisms underlying this process is critical to the clinical application of glucocorticoids for asthma therapy. After sensitization and challenge with ovalbumin (OVA), BALB/c mice were treated with inhaled budesonide and infected with Pseudomonas aeruginosa (P. aeruginosa). The number of viable bacteria in enflamed lungs was evaluated, and levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum were measured. A lung epithelial cell line was pretreated with budesonide. Levels of cathelicidin-related antimicrobial peptide (CRAMP) were measured by immunohistochemistry and western blot analysis. Intracellular bacteria were observed in lung epithelial cells. Results Inhaled budesonide enhanced lung infection in allergic mice exposed to P. aeruginosa and increased the number of viable bacteria in lung tissue. Higher levels of IL-4 and lower levels of IFN-γ were observed in the serum. Budesonide decreased the expression of CRAMP, increased the number of internalized P. aeruginosa in OVA-challenged mice and in lung epithelial cell lines. These data indicate that inhaled budesonide can suppress pulmonary antibacterial host defense by down-regulating CRAMP in allergic inflammation mice and in cells in vitro. Conclusions Inhaled budesonide suppressed pulmonary antibacterial host defense in an asthmatic mouse model and in lung epithelium cells in vitro. This effect was dependent on the down-regulation of CRAMP. PMID:23387852

A Novel Peptide to Treat Oral Mucositis Blocks Endothelial and Epithelial Cell Apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu Xiaoyan; Chen Peili; Sonis, Stephen T.

2012-07-01

Purpose: No effective agents currently exist to treat oral mucositis (OM) in patients receiving chemoradiation for the treatment of head-and-neck cancer. We identified a novel 21-amino acid peptide derived from antrum mucosal protein-18 that is cytoprotective, mitogenic, and motogenic in tissue culture and animal models of gastrointestinal epithelial cell injury. We examined whether administration of antrum mucosal protein peptide (AMP-p) could protect against and/or speed recovery from OM. Methods and Materials: OM was induced in established hamster models by a single dose of radiation, fractionated radiation, or fractionated radiation together with cisplatin to simulate conventional treatments of head-and-neck cancer. Results:more » Daily subcutaneous administration of AMP-p reduced the occurrence of ulceration and accelerated mucosal recovery in all three models. A delay in the onset of erythema after irradiation was observed, suggesting that a protective effect exists even before injury to mucosal epithelial cells occurs. To test this hypothesis, the effects of AMP-p on tumor necrosis factor-{alpha}-induced apoptosis were studied in an endothelial cell line (human dermal microvascular endothelial cells) as well as an epithelial cell line (human adult low-calcium, high-temperature keratinocytes; HaCaT) used to model the oral mucosa. AMP-p treatment, either before or after cell monolayers were exposed to tumor necrosis factor-{alpha}, protected against development of apoptosis in both cell types when assessed by annexin V and propidium iodide staining followed by flow cytometry or ligase-mediated polymerase chain reaction. Conclusions: These observations suggest that the ability of AMP-p to attenuate radiation-induced OM could be attributable, at least in part, to its antiapoptotic activity.« less

Metastatic prostate cancer-associated P62 inhibits autophagy flux and promotes epithelial to mesenchymal transition by sustaining the level of HDAC6.

PubMed

Jiang, Xianhan; Huang, Yiqiao; Liang, Xue; Jiang, Funeng; He, Yongzhong; Li, Tian; Xu, Guibin; Zhao, Haibo; Yang, Weiqing; Jiang, Ganggang; Su, Zhengming; Jiang, Lingke; Liu, Leyuan

2018-05-01

P62 (also named sequestosome-1, SQSTM1) is involved in autophagy regulation through multiple pathways. It interacts with autophagosomes-associated LC3-II and ubiquitinated protein aggregates to engulf the aggregates in autophagosomes, interacts with HDAC6 to inhibit its deacetylase activity to maintain the levels of acetylated α-tubulin and stabilities of microtubules to enhance autophagosome trafficking, and regulates autophagy initiation and cell survival. We performed immunohistochemistry staining of P62 in prostate tissues from prostate cancer patients and found that levels of P62 in patients with prostate adenocarcinomas (PCA) are significantly higher than those in patients with benign prostate hyperplasia (BPH). High levels of P62 predict high tumor grade and high intensity of metastasis. We created prostate cancer cell lines stably overexpressing P62 and then suppress the expression of P62 in the cell line stably overexpressing P62 with CRISPR technology. Cell proliferation assay with crystal violet, cell migration assay, cell invasion assay, Western blot analysis, and confocal fluorescent microscopy were conducted to test the impact of altered levels of P62 on the growth, migration, invasion, epithelial-to-mesenchymal transition, autophagy flux, HDAC6 activity, and microtubular acetylation of cancer cells. P62 increased the levels of HDAC6 and reduced the acetylation of α-tubulin and the stability of microtubules. Consequently, high levels of P62 caused a promotion of epithelial-to-mesenchymal transition in addition to an impairment of autophagy flux, and further led to an enhancement of proliferation, migration, and invasion of prostate cancer cells. P62 promotes metastasis of PCA by sustaining the level of HDAC6 to inhibit autophagy and promote epithelial-to-mesenchymal transition. © 2018 Wiley Periodicals, Inc.

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

PubMed Central

Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

2014-01-01

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53−/− mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53−/− mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers. PMID:24722541

Lymphangioma circumscriptum, angiokeratoma, or superficial vascular ectasia with epithelial hyperplasia?

PubMed

Katsoulas, Nikolaos; Tosios, Konstantinos I; Argyris, Prokopios; Koutlas, Ioannis G; Sklavounou, Alexandra

2014-08-01

We report a case of lymphangioma circumscriptum (cavernous lymphangioma with epithelial hyperplasia) in a 12-year-old girl, presenting as a papillary tumor on the right dorsal side of her tongue. Microscopic examination found cavernous vascular channels lined by a single layer of CD31(+), podoplanin-positive, CD34(-) endothelial cells that occupied the papillary lamina propria and were accompanied by epithelial hyperplasia. A review of the literature on oral vascular tumors with epithelial hyperplasia, namely, lymphangioma circumscriptum and angiokeratoma, provided information that draws into question the use of these terms. Copyright © 2014 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Booth, Brian W., E-mail: brbooth@clemson.edu; Institute for Biological Interfaces of Engineering, Clemson University, Clemson, SC 29634; Boulanger, Corinne A.

2010-02-01

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

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

PubMed

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

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D beta-geo (CDbetageo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDbetageo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG(-/-) mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro. Copyright 2009 Elsevier Inc. All rights reserved.

In vitro effect of phototherapy with low-intensity laser on HSV-1 and epithelial cells

NASA Astrophysics Data System (ADS)

Eduardo, Fernanda P.; Mehnert, Dolores U.; Monezi, Telma A.; Zezell, Denise M.; Schubert, Mark M.; Eduardo, Carlos P.; Marques, Márcia M.

2007-02-01

The effects of phototherapy on herpes lesions have been clinically demonstrated by either preventing the lesion formation or speeding their repair. The aim of this in vitro study was analyze the effect of phototherapy on epithelial cells and HSV-1 in culture. Cultures of HSV-1 and epithelial cells (Vero cell line) were used. The irradiations were done using a GaAlAs laser (660 e 780 nm, 4.0 mm2). One, two and three irradiations with 6 h-intervals were done. The experimental groups were: Control: non-irradiated; 660 nm and 3 J/cm2 (2.8 sec); 660 nm and 5 J/cm2 (3.8 sec); 780 nm and 3 J/cm2 (1.9 sec), and 780 nm and 5 J/cm2 (2.5 sec). The HSV-1 cytopatic effect and the cell viability of irradiated cultures and controls were analyzed in four different conditions: irradiation of non-infected epithelial cells; epithelial cells irradiated prior infection; virus irradiated prior infection; irradiation of HSV infected cells. The mitochondrial activity and cytopathic effects were assessed. The number of irradiations influenced the cell growth positively and proportionally, except for the 660 nm/ 3 J/cm2 group. Any variation in cytopathic effects was observed amongst the experimental groups. The viability of infected cells prior irradiation was significantly higher than that of non-irradiated cultures when 2 irradiations were done. Under the experimental conditions of this study we concluded that phototherapy is capable of enhancing epithelial cell growth and prolonging cell viability of HSV-1 infected cells. Positive benefits of phototherapy could be resultant from prolongation of infected cells viability, corroborating with host defenses.

Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines--do we have a proper model?

PubMed

Molenda, Natalia; Urbanova, Katarina; Weiser, Nelly; Kusche-Vihrog, Kristina; Günzel, Dorothee; Schillers, Hermann

2014-01-01

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.

Sodium Phenylbutyrate Inhibits Tumor Growth and the Epithelial-Mesenchymal Transition of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

PubMed

Qian, Kun; Sun, Laiyu; Zhou, Guoqing; Ge, Haixia; Meng, Yue; Li, Jingfen; Li, Xiao; Fang, Xinqiang

2018-05-01

Sodium phenylbutyrate (SPB) as a salt of 4-phenylbutyric acid (4-PBA) has been reported to be an ammonia scavenger, histone deacetylase inhibitor, and an endoplasmic reticulum stress inhibitor in various diseases, including neurological diseases, inflammatory disorders, and carcinogenesis. Although phenylbutyrate showed effective antitumor properties in many cancers, its role in oral squamous cell carcinoma (OSCC) remains further characterized. Thus, the OSCC cell lines CAL27, HSC3, and SCC4 were treated with a series of doses of SPB for different times. The IC 50 of three cell lines for SPB was determined to be 4.0, 3.7, and 3.0 mM. The CCK-8 assay indicated that the treatment of SPB induced continuous inhibition of cell vitality of three cell lines. Apoptosis was assessed by Hoechst assay that showed that SPB could significantly promote cell apoptosis. Moreover, the apoptosis-related pathway was analyzed, and the results showed that the expression of antiapoptosis factor BCL-2 was downregulated by SPB but the cleavage of caspase-3 was increased. Meanwhile, it was found that SPB also impaired the migration and invasion of OSCC cells in vitro. Mechanistically, the transforming growth factor-β (TGFB) related epithelial-mesenchymal transition (EMT) was inhibited by SPB with decreased mesenchymal marker N-cadherin and increased epithelial marker E-cadherin. Furthermore, the antitumor effect of SPB in vivo was also demonstrated. The administration of SPB induced remarkably tumor regression with decreased tumor volume, and the TGFB level and EMT phenotype in vivo were also inhibited. These data demonstrated that the treatment of SPB could function as antitumor therapeutics for OSCC.

Ultramicroscopic examination of the ovine tonsillar epithelia.

PubMed

Casteleyn, Christophe; Cornelissen, Maria; Simoens, Paul; Van den Broeck, Wim

2010-05-01

As solid morphological knowledge of ovine tonsillar epithelia might contribute to a better understanding of the pathogenesis of several diseases including prion diseases, the epithelia of all tonsils of 7 one-year-old Texel sheep were examined using scanning and transmission electron microscopy. Major parts of the pharyngeal and tubal tonsils were covered by pseudostratified columnar ciliated epithelia that were interrupted by patches of epithelium containing cells with densely packed microfolds or microvilli, and cells with both microvilli and cilia. Smaller parts were covered by either flattened polygonal cells with densely packed microvilli or microfolds, squamous epithelial cells, or patches of reticular epithelium. The palatine and paraepiglottic tonsils were mainly lined by squamous epithelial cells with apical microplicae or short knobs. Additionally, regions of reticular epithelium containing epithelial cells with apical microvilli were seen. The lingual tonsil was uniformly covered by a keratinized squamous epithelium and devoid of microvillous cells and patches of reticular epithelium. The rostral half of the tonsil of the soft palate was lined by a pseudostratified columnar ciliated epithelium with characteristics of the pharyngeal and tubal tonsils. The epithelium of the caudal part resembled the epithelia of the palatine and paraepiglottic tonsils. Putative M cells, mainly characterized by apical microvilli or microfolds and a close association with lymphoid cells, seem manifestly present on the nasopharyngeal tonsils. The reticular epithelium of the palatine and paraepiglottic tonsils also harbor cells with small apical microvilli. The exact nature of these presumptive M cells should, however, be elucidated in functional studies.

Inhibition of gamma-secretase in Notch1 signaling pathway as a novel treatment for ovarian cancer.

PubMed

Feng, Zhaoyi; Xu, Wandong; Zhang, Chenguang; Liu, Mengran; Wen, Hongwu

2017-01-31

Epithelial ovarian cancer (EOC) is the leading cause of death for gynecological cancer. Most patients are not diagnosed until the cancer is at an advanced stage with poor prognosis. Notch1 signaling pathway plays an oncogenic role in EOC. There have been few studies on enzymatic activity of γ-secretase and the mechanism of how γ-secretase inhibitor works on cancer cell. Here, we show that Jagged1 and NICD were highly expressed in ovarian carcinoma. The expressions of Notch1, Jagged1 and NICD in Notch1 pathway did not correlate with outcome in ovarian cancer. The enzymatic activity of γ-secretase in ovarian cancer cell lines SKOV3, CAOV3 and ES2 is significantly higher than in normal ovarian epithelial cell line T29. DAPT (a γ-secretase inhibitor) reduced the enzymatic activity of γ-secretase, inhibited the proliferation, and increased the apoptosis in ovarian cancer cell lines. Hence, γ-secretase inhibitor may become a highly promising novel therapeutic strategy against ovarian cancer in the field of precision medicine.

Bioengineered 2'-fucosyllactose and 3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and enteric pathogens to human intestinal and respiratory cell lines.

PubMed

Weichert, Stefan; Jennewein, Stefan; Hüfner, Eric; Weiss, Christel; Borkowski, Julia; Putze, Johannes; Schroten, Horst

2013-10-01

Human milk oligosaccharides help to prevent infectious diseases in breastfed infants. Larger scale testing, particularly in animal models and human clinical studies, is still limited due to shortened availability of more complex oligosaccharides. The purpose of this study was to evaluate 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) synthesized by whole-cell biocatalysis for their biological activity in vitro. Therefore, we have tested these oligosaccharides for their inhibitory potential of pathogen adhesion in two different human epithelial cell lines. 2'-FL could inhibit adhesion of Campylobacter jejuni, enteropathogenic Escherichia coli, Salmonella enterica serovar fyris, and Pseudomonas aeruginosa to the intestinal human cell line Caco-2 (reduction of 26%, 18%, 12%, and 17%, respectively), as could be shown for 3-FL (enteropathogenic E coli 29%, P aeruginosa 26%). Furthermore, adherence of P aeruginosa to the human respiratory epithelial cell line A549 was significantly inhibited by 2'-FL and 3-FL (reduction of 24% and 23%, respectively). These results confirm the biological and functional activity of biotechnologically synthesized human milk oligosaccharides. Mass-tailored human milk oligosaccharides could be used in the future to supplement infant formula ingredients or as preventatives to reduce the impact of infectious diseases. © 2013 Elsevier Inc. All rights reserved.

Changes in barrier function of a model intestinal epithelium by intraepithelial lymphocytes require new protein synthesis by epithelial cells.

PubMed Central

Taylor, C T; Murphy, A; Kelleher, D; Baird, A W

1997-01-01

BACKGROUND: Elements of the mucosal immune system may play an important part in regulating epithelial barrier function in the intestinal tract. Intraepithelial lymphocytes (IELs) represent a subtype of immunocyte which is strategically placed to regulate epithelial function at most mucosal sites. AIMS AND METHODS: An IEL derived cell line (SC1) was used to examine its effects on the model epithelium T84--a tumour derived cell line which retains the phenotype of colonic crypt cells. Transepithelial electrical resistance (TER) was used as a marker of epithelial integrity. RESULTS: Coculture of T84 cells with SC1 produced a significant fall in TER as did exposure of T84 monolayers to IEL derived supernatant. Recombinant interferon-gamma (rIFN gamma) also reduced TER in T84 monolayers. Cycloheximide prevented the effects of IEL supernatant and of rIFN gamma on TER. The fall in TER in response to rIFN gamma was attenuated by blocking antibodies, which did not alter the fall in resistance induced by IEL supernatant. Fractions of IEL supernatant, separated on the basis of size, evoked temporally distinct changes in TER. Ultrastructural studies support the hypothesis that the slow onset but severe fall in TER indicates catastrophic effects on the monolayer. The more rapid onset fall in TER was not associated with gross changes in monolayer morphology. Reduction of TER by IEL supernatant was not influenced by inhibitors of tyrosine phosphatase or of protein kinase C. Although herbimycin did reduce the rapid onset change in TER, the tyrosine kinase inhibitor genistein did not alter responses to IEL supernatant. CONCLUSIONS: Mucosal T cells may influence barrier function by a process involving new protein synthesis by epithelial cells. This model may have relevance in some inflammatory conditions of the gastrointestinal tract. Images PMID:9203943

[Ultrastructure of the digestive system in Dermatophagoides farinae (Acariformes:Pyroglyphidae)].

PubMed

Wang, Yue-Ming; Liu, Xiao-Yu; Jiang, Cong-Li; Huang, Li-Nian; Sun, Xin; Liu, Zhi-Gang

2013-12-01

Fifty living mites (Dermatophagoides farinae) were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, embedded in embedding medium. The ultrastructure of the digestive tract in D. farinae was observed by serial ultrathin sections with a transmission electron microscope. The alimentary canal of D. farinae consists of the cuticle-lined foregut and hindgut separated by a microvilli-lined midgut (anterior midgut, posterior midgut). There are different types of epithelial cells in the anterior midgut The microvilli of epithelial cells in posterior midgut are longer than that of the anterior midgut In posterior midgut, the food bolus is surrounded by the peritrophic membrane. The midgut is the main site of digestion and absorption.

Expression of membrane-associated mucins MUC1 and MUC4 in major human salivary glands.

PubMed

Liu, Bing; Lague, Jessica R; Nunes, David P; Toselli, Paul; Oppenheim, Frank G; Soares, Rodrigo V; Troxler, Robert F; Offner, Gwynneth D

2002-06-01

Mucins are high molecular weight glycoproteins secreted by salivary glands and epithelial cells lining the digestive, respiratory, and reproductive tracts. These glycoproteins, encoded in at least 13 distinct human genes, can be subdivided into gel-forming and membrane-associated forms. The gel-forming mucin MUC5B is secreted by mucous acinar cells in major and minor salivary glands, but little is known about the expression pattern of membrane-associated mucins. In this study, RT-PCR and Northern blotting demonstrated the presence of transcripts for MUC1 and MUC4 in both parotid and submandibular glands, and in situ hybridization localized these transcripts to epithelial cells lining striated and excretory ducts and in some serous acinar cells. The same cellular distribution was observed by immunohistochemistry. Soluble forms of both mucins were detected in parotid secretion after immunoprecipitation with mucin-specific antibodies. These studies have shown that membrane-associated mucins are produced in both parotid and submandibular glands and that they are expressed in different cell types than gel-forming mucins. Although the function of these mucins in the oral cavity remains to be elucidated, it is possible that they both contribute to the epithelial protective mucin layer and act as receptors initiating one or more intracellular signal transduction pathways.

Ontological representation, integration, and analysis of LINCS cell line cells and their cellular responses.

PubMed

Ong, Edison; Xie, Jiangan; Ni, Zhaohui; Liu, Qingping; Sarntivijai, Sirarat; Lin, Yu; Cooper, Daniel; Terryn, Raymond; Stathias, Vasileios; Chung, Caty; Schürer, Stephan; He, Yongqun

2017-12-21

Aiming to understand cellular responses to different perturbations, the NIH Common Fund Library of Integrated Network-based Cellular Signatures (LINCS) program involves many institutes and laboratories working on over a thousand cell lines. The community-based Cell Line Ontology (CLO) is selected as the default ontology for LINCS cell line representation and integration. CLO has consistently represented all 1097 LINCS cell lines and included information extracted from the LINCS Data Portal and ChEMBL. Using MCF 10A cell line cells as an example, we demonstrated how to ontologically model LINCS cellular signatures such as their non-tumorigenic epithelial cell type, three-dimensional growth, latrunculin-A-induced actin depolymerization and apoptosis, and cell line transfection. A CLO subset view of LINCS cell lines, named LINCS-CLOview, was generated to support systematic LINCS cell line analysis and queries. In summary, LINCS cell lines are currently associated with 43 cell types, 131 tissues and organs, and 121 cancer types. The LINCS-CLO view information can be queried using SPARQL scripts. CLO was used to support ontological representation, integration, and analysis of over a thousand LINCS cell line cells and their cellular responses.

18β-glycyrrhetinic acid potentiates Hsp90 inhibition-induced apoptosis in human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial pathway.

PubMed

Yang, Jae Chon; Myung, Soon Chul; Kim, Wonyong; Lee, Chung Soo

2012-11-01

The Hsp90 inhibition has been shown to induce apoptosis in various cancer cells. The licorice compounds may enhance the anti-cancer drug effect. However, effect of the licorice compounds on the Hsp90 inhibition-induced apoptosis in ovarian cancer cells has not been studied. To assess the ability of 18β-glycyrrhetinic acid to promote apoptosis, we examined whether 18β-glycyrrhetinic acid potentiated the Hsp90 inhibitor-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Radicicol and geldanamycin induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels, an increase in Bax levels, the mitochondrial transmembrane potential loss, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1, and an increase in the tumor suppressor p53 levels. 18β-Glycyrrhetinic acid enhanced Hsp90 inhibitor-induced apoptosis-related protein activation, nuclear damage, and cell death. The results suggest that 18β-glycyrrhetinic acid may potentiate the Hsp90 inhibition-induced apoptosis in ovarian carcinoma cell lines via the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated cell death pathway, leading to activation of caspases. Combination of Hsp90 inhibitors and 18β-glycyrrhetinic acid may confer a benefit in the treatment of epithelial ovarian adenocarcinoma.

Probiotics promote endocytic allergen degradation in gut epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Chun-Hua; Liu, Zhi-Qiang; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON

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 barriermore » 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.« less

In vitro model alveoli from photodegradable microsphere templates†

PubMed Central

Lewis, Katherine J. R.; Tibbitt, Mark W.; Zhao, Yi; Branchfield, Kelsey; Sun, Xin; Balasubramaniam, Vivek; Anseth, Kristi S.

2016-01-01

Recreating the 3D cyst-like architecture of the alveolar epithelium in vitro has been challenging to achieve in a controlled fashion with primary lung epithelial cells. Here, we demonstrate model alveoli formed within a tunable synthetic biomaterial platform using photodegradable microspheres as templates to create physiologically relevant, cyst structures. Poly(ethylene glycol) (PEG)-based hydrogels were polymerized in suspension to form microspheres on the order of 120 μm in diameter. The gel chemistry was designed to allow erosion of the microspheres with cytocompatible light doses (≤15 min exposure to 10 mW cm−2 of 365 nm light) via cleavage of a photolabile nitrobenzyl ether crosslinker. Epithelial cells were incubated with intact microspheres, modified with adhesive peptide sequences to facilitate cellular attachment to and proliferation on the surface. A tumor-derived alveolar epithelial cell line, A549, completely covered the microspheres after only 24 hours, whereas primary mouse alveolar epithelial type II (ATII) cells took ~3 days. The cell-laden microsphere structures were embedded within a second hydrogel formulation at user defined densities; the microsphere templates were subsequently removed with light to render hollow epithelial cysts that were cultured for an additional 6 days. The resulting primary cysts stained positive for cell–cell junction proteins (β-catenin and ZO-1), indicating the formation of a functional epithelial layer. Typically, primary ATII cells differentiated in culture to the alveolar epithelial type I (ATI) phenotype; however, each cyst contained ~1–5 cells that stained positive for an ATII marker (surfactant protein C), which is consistent with ATII cell numbers in native mouse alveoli. This biomaterial-templated alveoli culture system should be useful for future experiments to study lung development and disease progression, and is ideally suited for co-culture experiments where pulmonary fibroblasts or endothelial cells could be presented in the hydrogel surrounding the epithelial cysts. PMID:26221842

Expanding and characterizing esophageal epithelial cells obtained from children with eosinophilic esophagitis.

PubMed

Sayej, Wael N; Foster, Christopher; Jensen, Todd; Chatfield, Sydney; Finck, Christine

2018-06-12

The role of epithelial cells in eosinophilic esophagitis (EoE) is not well understood. In this study, our aim was to isolate, culture, and expand esophageal epithelial cells obtained from patients with or without EoE and characterize differences observed over time in culture. Biopsies were obtained at the time of endoscopy from children with EoE or suspected to have EoE. We established patient-derived esophageal epithelial cell (PDEEC) lines utilizing conditional reprogramming methods. We determined integrin profiles, gene expression, MHC class II expression, and reactivity to antigen stimulation. The PDEECs were found to maintain their phenotype over several passages. There were differences in integrin profiles and gene expression levels in EoE-Active compared to normal controls and EoE-Remission patients. Once stimulated with antigens, PDEECs express MHC class II molecules on their surface, and when co-cultured with autologous T-cells, there is increased IL-6 and TNF-α secretion in EoE-Active patients vs. controls. We are able to isolate, culture, and expand esophageal epithelial cells from pediatric patients with and without EoE. Once stimulated with antigens, these cells express MHC class II molecules and behave as non-professional antigen-presenting cells. This method will help us in developing an ex vivo, individualized, patient-specific model for diagnostic testing for causative antigens.

Effects of cyclosporin A on a kidney epithelial cell line (LLC-PK1).

PubMed

Becker, G M; Gandolfi, A J; Nagle, R B

1987-05-01

Cyclosporin A (CSA), a potent immunosuppressant with the adverse side effect of nephrotoxicity, inhibited cell growth of pig kidney tubule cells (LLC-PK1) in culture. CSA (10(-5) M) also induced intense cytoplasmic vacuolation and the formation of dense granules. At the same concentration an analogue of CSA, cyclosporin G, had much less effect. This cell line may prove useful for revealing the mechanism of CSA-nephrotoxicity and testing the nephrotoxic potential of new analogues of cyclosporine.

Detection of increased choline compounds with proton nuclear magnetic resonance spectroscopy subsequent to malignant transformation of human prostatic epithelial cells.

PubMed

Ackerstaff, E; Pflug, B R; Nelson, J B; Bhujwalla, Z M

2001-05-01

In this study, a panel of normal human prostate cells (HPCs) and tumor cells derived from metastases were studied by (1)H NMR spectroscopy to determine whether the malignant transformation of HPCs results in the elevation of choline compounds. Although an elevated choline signal has been observed previously in clinical studies, the contribution of the different Cho compounds to this elevation, as well as their quantification, has not been established until now. Here we have shown that HPCs derived from metastases exhibit significantly higher phosphocholine as well as glycerophosphocholine levels compared with normal prostate epithelial and stromal cells. Thus the elevation of the choline peak observed clinically in prostate cancer is attributable to an alteration of phospholipid metabolism and not simply to increased cell density, doubling time, or other nonspecific effects. Androgen deprivation of the androgen receptor-positive cell lines resulted in a significant increase of choline compounds after chronic androgen deprivation of the LNCaP cell line and in a decrease of choline compounds after a more acute androgen deprivation of the LAPC-4 cell line. These data strongly support the use of proton magnetic resonance spectroscopic imaging to detect the presence of prostate cancer for diagnosis, to detect response subsequent to androgen ablation therapy, and to detect recurrence.

The effect of mechanical extension stimulation combined with epithelial cell sorting on outcomes of implanted tissue-engineered muscular urethras.

PubMed

Fu, Qiang; Deng, Chen-Liang; Zhao, Ren-Yan; Wang, Ying; Cao, Yilin

2014-01-01

Urethral defects are common and frequent disorders and are difficult to treat. Simple natural or synthetic materials do not provide a satisfactory curative solution for long urethral defects, and urethroplasty with large areas of autologous tissues is limited and might interfere with wound healing. In this study, adipose-derived stem cells were used. These cells can be derived from a wide range of sources, have extensive expansion capability, and were combined with oral mucosal epithelial cells to solve the problem of finding seeding cell sources for producing the tissue-engineered urethras. We also used the synthetic biodegradable polymer poly-glycolic acid (PGA) as a scaffold material to overcome issues such as potential pathogen infections derived from natural materials (such as de-vascular stents or animal-derived collagen) and differing diameters. Furthermore, we used a bioreactor to construct a tissue-engineered epithelial-muscular lumen with a double-layer structure (the epithelial lining and the muscle layer). Through these steps, we used an epithelial-muscular lumen built in vitro to repair defects in a canine urethral defect model (1 cm). Canine urethral reconstruction was successfully achieved based on image analysis and histological techniques at different time points. This study provides a basis for the clinical application of tissue engineering of an epithelial-muscular lumen. Copyright © 2013 Elsevier Ltd. All rights reserved.

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

PubMed Central

Wynford-Thomas, D; Bond, J A; Wyllie, F S; Burns, J S; Williams, E D; Jones, T; Sheer, D; Lemoine, N R

1990-01-01

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types. Images PMID:1697930

Human bronchial epithelial cells injury and cytokine production induced by Tityus serrulatus scorpion venom: An in vitro study.

PubMed

Rigoni, Vera Lucia Silva; Kwasniewski, Fabio H; Vieira, Rodolfo Paula; Linhares, Ingrid Sestrem; da Silva, Joelmir Lucena Veiga; Nogueira-Pedro, Amanda; Zamuner, Stella Regina

2016-09-15

Tityus serrulatus is the scorpion specie responsible for the majority of scorpion sting accidents in Brazil. Symptoms of envenomation by Tityus serrulatus range from local pain to severe systemic reactions such as cardiac dysfunction and pulmonary edema. Thus, this study has evaluated the participation of bronchial epithelial cells in the pulmonary effects of Tityus serrulatus scorpion venom (Tsv). Human bronchial epithelial cell line BEAS-2B were utilized as a model target and were incubated with Tsv (10 or 50 μg/mL) for 1, 3, 6 and 24 h. Effects on cellular response of venom-induce cytotoxicity were examined including cell viability, cell integrity, cell morphology, apoptosis/necrosis as well as cell activation through the release of pro-inflammatory cytokines IL-1β, IL-6 and IL-8. Tsv caused a decrease in cell viability at 10 and 50 μg/mL, which was confirmed by lactate dehydrogenase (LDH) measurement. Flow cytometry analyses revealed necrosis as the main cell death pathway caused by Tsv. Furthermore, Tsv induced the release of IL-1β, IL-6 and IL-8. Altogether, these results demonstrate that Tsv induces cytotoxic effects on bronchial epithelial cells, involving necrosis and release of pro-inflammatory cytokines, suggesting that bronchial epithelial cells may play a role in the pulmonary injury caused by Tsv. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vivo biomarker expression patterns are preserved in 3D cultures of Prostate Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Windus, Louisa C.E.; Kiss, Debra L.; Glover, Tristan

2012-11-15

Here we report that Prostate Cancer (PCa) cell-lines DU145, PC3, LNCaP and RWPE-1 grown in 3D matrices in contrast to conventional 2D monolayers, display distinct differences in cell morphology, proliferation and expression of important biomarker proteins associated with cancer progression. Consistent with in vivo growth rates, in 3D cultures, all PCa cell-lines were found to proliferate at significantly lower rates in comparison to their 2D counterparts. Moreover, when grown in a 3D matrix, metastatic PC3 cell-lines were found to mimic more precisely protein expression patterns of metastatic tumour formation as found in vivo. In comparison to the prostate epithelial cell-linemore » RWPE-1, metastatic PC3 cell-lines exhibited a down-regulation of E-cadherin and {alpha}6 integrin expression and an up-regulation of N-cadherin, Vimentin and {beta}1 integrin expression and re-expressed non-transcriptionally active AR. In comparison to the non-invasive LNCaP cell-lines, PC3 cells were found to have an up-regulation of chemokine receptor CXCR4, consistent with a metastatic phenotype. In 2D cultures, there was little distinction in protein expression between metastatic, non-invasive and epithelial cells. These results suggest that 3D cultures are more representative of in vivo morphology and may serve as a more biologically relevant model in the drug discovery pipeline. -- Highlights: Black-Right-Pointing-Pointer We developed and optimised 3D culturing techniques for Prostate Cancer cell-lines. Black-Right-Pointing-Pointer We investigated biomarker expression in 2D versus 3D culture techniques. Black-Right-Pointing-Pointer Metastatic PC3 cells re-expressed non-transcriptionally active androgen receptor. Black-Right-Pointing-Pointer Metastatic PCa cell lines retain in vivo-like antigenic profiles in 3D cultures.« less

MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype

PubMed Central

Kietzmann, Leonie; Guhr, Sebastian S.O.; Meyer, Tobias N.; Ni, Lan; Sachs, Marlies; Panzer, Ulf; Stahl, Rolf A.K.; Saleem, Moin A.; Kerjaschki, Dontscho; Gebeshuber, Christoph A.

2015-01-01

Parietal epithelial cells have been identified as potential progenitor cells in glomerular regeneration, but the molecular mechanisms underlying this process are not fully defined. Here, we established an immortalized polyclonal human parietal epithelial cell (hPEC) line from naive human Bowman’s capsule cells isolated by mechanical microdissection. These hPECs expressed high levels of PEC-specific proteins and microRNA-193a (miR-193a), a suppressor of podocyte differentiation through downregulation of Wilms’ tumor 1 in mice. We then investigated the function of miR-193a in the establishment of podocyte and PEC identity and determined whether inhibition of miR-193a influences the behavior of PECs in glomerular disease. After stable knockdown of miR-193a, hPECs adopted a podocyte-like morphology and marker expression, with decreased expression levels of PEC markers. In mice, inhibition of miR-193a by complementary locked nucleic acids resulted in an upregulation of the podocyte proteins synaptopodin and Wilms’ tumor 1. Conversely, overexpression of miR-193a in vivo resulted in the upregulation of PEC markers and the loss of podocyte markers in isolated glomeruli. Inhibition of miR-193a in a mouse model of nephrotoxic nephritis resulted in reduced crescent formation and decreased proteinuria. Together, these results show the establishment of a human PEC line and suggest that miR-193a functions as a master switch, such that glomerular epithelial cells with high levels of miR-193a adopt a PEC phenotype and cells with low levels of miR-193a adopt a podocyte phenotype. miR-193a–mediated maintenance of PECs in an undifferentiated reactive state might be a prerequisite for PEC proliferation and migration in crescent formation. PMID:25270065

Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals.

PubMed

Jensen, Helene H; Pedersen, Hans N; Stenkjær, Eva; Pedersen, Gitte A; Login, Frédéric H; Nejsum, Lene N

2015-01-01

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation.

Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals

PubMed Central

Jensen, Helene H.; Pedersen, Hans N.; Stenkjær, Eva; Pedersen, Gitte A.; Login, Frédéric H.; Nejsum, Lene N.

2015-01-01

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation. PMID:26536015

Biologic properties of the thymocyte-activating factor (CETAF) produced by a rabbit corneal cell line (SIRC).

PubMed

Grabner, G; Luger, T A; Luger, B M; Smolin, G; Oh, J O

1983-05-01

Rabbit corneal epithelial cell cultures produce a cytokine (CETAF) that greatly enhances the proliferation of C3H/HeJ mouse thymocytes. The rabbit corneal cell line SIRC was used to generate CETAF activity in the culture supernatant. CETAF was then partially purified by Sephacryl S-200 gel filtration, where peaks of activity eluted in a molecular weight range of 95,000-55,000 (CETAF I) and 30,000-15,000 (CETAF II). Similar to the epidermal cell-derived thymocyte-activating factor (ETAF), CETAF (I and II) stimulated the growth of a human dermal fibroblast line (CRL 1445) in a dose-dependent manner, but failed to enhance the proliferation of an Interleukin 2 (IL 2)-dependent T-cell line (CT 6). Although CETAF did not exhibit any IL 2 activity, it clearly enhanced the IL 2 production by C3H/HeJ mouse splenocytes stimulated with suboptimal doses of lectins. Crude SIRC supernatants as well as the partially purified CETAF preparations showed a marked inhibition of polymorphonuclear neutrophil migration at high concentrations, but were significantly chemotactic when diluted samples were tested. CETAF release by SIRC cells was increased by stimulation with mitomycin C, phorbolmyristate acetate, hydroxyurea, silica, lipopolysaccaride B, and when the cells were cultured under serum-free conditions. These observations suggest that corneal epithelial cells may not only interact with the immune system in a way similar to keratinocytes, but may also stimulate corneal stromal cell through the production of CETAF.

Expression of Ki-67 in odontogenic cysts: A comparative study between odontogenic keratocysts, radicular cysts and dentigerous cysts

PubMed Central

Modi, Tapan G; Chalishazar, Monali; Kumar, Malay

2018-01-01

Introduction and Objectives: Odontogenic cysts are the most common cysts of the jaws and are formed from the remnants of the odontogenic apparatus. Among these odontogenic cysts, radicular cysts (RCs) (about 60% of all diagnosed jaw cysts), dentigerous cysts (DCs) (16.6% of all jaw cysts) and odontogenic keratocysts (OKCs) (11.2% of all developmental odontogenic cysts) are the most common. The behavior of any lesion is generally reflected by its growth potential. Growth potential is determined by measuring the cell proliferative activity. The cell proliferative activity is measured by various methods among which immunohistochemistry (IHC) is the commonly used technique. Most of the IHC studies on cell proliferation have been based on antibodies such as Ki-67 and proliferating cell nuclear antigen. Materials and Method: In the present study, the total sample size comprised of 45 cases of odontogenic cysts, with 15 cases each of OKC, RC and DC. Here, an attempt is made to study immunohistochemical (streptavidin-biotin detection system HRP-DAB) method to assess the expression of Ki-67 in different layers of the epithelial lining of OKCs, RCs and DCs. Observations and Results: Ki-67 positive cells were highest in epithelium of OKC as compared to DC and RC. Conclusion: The increased Ki-67 labeling index and its expression in suprabasal cell layers of epithelial lining in OKC and its correlation with suprabasal cell layers of epithelial lining in DC and RC could contribute toward its clinically aggressive behavior. OKC is of more significance to the oral pathologist and oral surgeon because of its specific histopathological features, high recurrence rate and aggressive behavior. PMID:29731577

Exosomes as mediators of platinum resistance in ovarian cancer

PubMed Central

Crow, Jennifer; Atay, Safinur; Banskota, Samagya; Artale, Brittany; Schmitt, Sarah; Godwin, Andrew K

2017-01-01

Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells. PMID:28060758

Procyanidin-rich extract of natural cocoa powder causes ROS-mediated caspase-3 dependent apoptosis and reduction of pro-MMP-2 in epithelial ovarian carcinoma cell lines.

PubMed

Taparia, Shruti Sanjay; Khanna, Aparna

2016-10-01

Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease in invasive potential of the cells. Apoptosis and MMP2 downregulation appear to be linked to the increase in intracellular ROS levels, caused due to the prooxidant effect of cocoa procyanidin extract. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

PubMed

Kniss, Douglas A; Summerfield, Taryn L

2014-08-01

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

Grain dust induces IL-8 production from bronchial epithelial cells: the effect of dexamethasone on IL-8 production.

PubMed

Park, H S; Suh, J H; Kim, H Y; Kwon, O J; Choi, D C

1999-04-01

Recent publications have suggested an active participation of neutrophils to induce bronchoconstriction after inhalation of grain dust (GD). To further understand the role of neutrophils in the pathogenesis of GD-induced asthma, this investigation was designed to determine whether human bronchial epithelial cells could produce IL-8 production and to observe the effect of dexamethasone on IL-8 production. We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four concentrations (1 to 200 microg/mL) of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant, which was derived from the culture of PBMC from a GD-induced asthmatic subject under the exposure to 10 microg/mL of GD, and compared with those cultured without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. To evaluate the effect of dexamethasone on IL-8 production, four concentrations (5 to 5000 ng/mL) of dexamethasone were pre-incubated for 24 hours and the same experiments were repeated. There was significant production of IL-8 from bronchial epithelial cells with additions of GD in a dose-dependent manner (P < .05), which was significantly augmented with additions of PBMC supernatant (P < .05) at each concentration. Compared with the untreated sample, pretreatment of dexamethasone could induced a remarkable inhibitions (15% to 55%) of IL-8 production from bronchial epithelial cells in a dose-dependent manner. These results suggest that IL-8 production from bronchial epithelial cells may contribute to neutrophil recruitment occurring in GD-induced airway inflammation. The downregulation of IL-8 production by dexamethasone from bronchial epithelial cells may contribute to the efficacy of this compound in reducing cellular infiltration and ultimately to its anti-inflammatory property.

Alpha-Tocopherol alters transcription activities that modulate tumor necrosis factor alpha (TNF-¿)-induced inflammatory response in bovine cells

USDA-ARS?s Scientific Manuscript database

To further investigate the potential role of '-tocopherol in maintaining immuno-homeostasis in bovine cells (Madin-Darby bovine kidney epithelial cell line), we undertook in vitro experiments using recombinant TNF-a as an immuno-stimulant to simulate inflammation response in cells with and without '...

Hyperspectral Imaging, Flow cytometry and Microscopic Morphology of Silver Nanoparticle within Cells

EPA Science Inventory

The ability to detect and track silver nanoparticles (AgNP) that enter cells is important to understand the potential biological and toxicological actions of AgNP. The uptake and fate in cells of four different types of AgNP was studied in a retinal pigment epithelial cell line ...

CARDIAC-LIKE OSCILLATION IN LIVER STEM CELLS INDUCE THEIR ACQUISITION OF CARDIAC PHENOTYPE

EPA Science Inventory

We examined in a cardiac microenvironment the plasticity of a liver stem cell line (WB F344) generated from a cloned, single, non-parenchymal epithelial cell from a normal adult male rat. Our previous studies suggested that WB F344 cells acquire a cardiac phenotype in the absenc...

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lasalvia, Maria; Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari; Castellani, Stefano

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

TPA induces a block of differentiation and increases the susceptibility to neoplastic transformation of a rat thyroid epithelial cell line.

PubMed

Portella, G; Vitagliano, D; Li, Z; Sferratore, F; Santoro, M; Vecchio, G; Fusco, A

1998-01-01

The PC Cl 3 cell line is a well-characterized epithelial cell line of rat thyroid origin. This cell line retains in vitro the typical markers of thyroid differentiation: thyroglobulin (TG) synthesis and secretion, iodide uptake, thyroperoxidase (TPO) expression, and dependency on TSH for growth. Although the differentiated phenotype of thyroid cells has been relatively well described, the molecular mechanisms that regulate both differentiation and neoplastic transformation of thyroid cells still need to be investigated in detail. Protein kinase C (PKC), the target of tetradecanoylphorbol acetate (TPA), regulates growth and differentiation of several cell types. Here we show that treatment of PC Cl 3 cells with TPA induces an acute block of thyroid differentiation. TPA-treated PC Cl 3 cells are unable to trap iodide and the expression levels of thyroglobulin, TSH receptor, and TPO genes are drastically reduced by TPA treatment. This differentiation block is not caused by a reduced expression of one of the master genes of thyroid differentiation, the thyroid transcription factor 1 (TTF-1). TPA-treated PC Cl 3 cells display an increased growth rate indicating that, in addition to the differentiation block, TPA also significantly affects the growth regulation of thyroid cells. Finally, TPA treatment dramatically increases the number of transformation foci induced in PC Cl 3 cells by retroviruses carrying v-Ki-ras, v-Ha-ras, and v-mos oncogenes. These findings support the notion that the PKC pathway can influence proliferation, differentiation, and neoplastic transformation of thyroid cells in culture.

Development, validation and implementation of an in vitro model for the study of metabolic and immune function in normal and inflamed human colonic epithelium.

PubMed

Pedersen, Gitte

2015-01-01

Ulcerative colitis (UC) and Crohn's disease (CD), collectively referred to as inflammatory bowel disease (IBD), are chronic immune disorders affecting the gastrointestinal tract. The aetiology of IBD remains an enigma, but increasing evidence suggests that the development of IBD may be triggered by a disturbance in the balance between gut commensal bacteria and host response in the intestinal mucosa. It is now known that epithelial cells have the capacity to secrete and respond to a range of immunological mediators and this suggests that these cells play a prominent role in the pathogenesis of IBD. Current knowledge about the intestinal epithelium has mainly been obtained using models based on animal cells, transformed human intestinal cell lines and isolated cells from resected colonic bowel segments. Species difference, malignant origin and confounders related to surgery, obviously make these cell models however less applicable for patophysiological studies. Consequently, there was a clear need for models of representative intestinal epithelial cells that would allow functional and dynamic studies of the differentiated human colonic epithelium in vitro. The primary purpose of this thesis was to explore and validate the optimal conditions for establishing a model based on short-term cultures of human colonic epithelial cells obtained from endoscopical biopsies. The cell cultures were accordingly used to describe the interplay between proinflammatory cytokines and colonic epithelium, with focus on alterations in viability, butyrate metabolism and secretion of a chemokine and metalloproteinases (MMP). Finally, the model was used to characterize expression and activation of receptors like toll like receptor (TLR)9 and peroxisome activated proliferators (PPAR)- known to be important players in regulation of innate and adaptive immune responses in human colonic epithelium. The results showed that it is possible to establish short-term cultures of representative, viable human colonic epithelial cells from endoscopic mucosal biopsies of patients with IBD. Short-time isolation by EGTA/EDTA from colonic biopsies allowed establishment of small scale cultures of epithelial cells which were viable and metabolic active for up to 48 hours in vitro. The cell model preserved important cellular metabolic and immunological functions of the human colonic epithelium, including the ability to oxidate butyrate, detoxificate phenolic compounds and secrete the chemokine interleukin (IL)-8 in vitro. Tumour necrosis factor (TNF)-α and interferon (IFN)-γ are pro-inflammatory cytokines, which are present in increased amounts in inflamed colonic mucosa. The precise mechanisms of cytokine-mediated mucosal injury are unknown, but one might be that TNF-α and IFN-γ directly impair epithelial cell function similar to effects seen on distinct target cells in other autoimmune diseases. Using the model, both cytokines were found directly to impair the viability of colonic epithelial cells and to induce secretion of IL-8 in vitro. Interestingly, the cells from inflamed IBD mucosa were less sensitive to cytokine-induced damage, which suggests that an intrinsic defense mechanism is triggered in these cells, perhaps as a result of exposure to toxic luminal factors or high local cytokine levels in vivo. TNF-α and IFN-γ may also be involved in regulation of intestinal inflammation through stimulation of MMP expression and proteolytic activity. We found that colonic epithelial cells express a range of MMPs and moreover that expression of distinct MMPs is increased in cells from inflamed IBD mucosa. Using a functional peptide cleavage assay it was shown that epithelial cells secreted proteolytic active enzymes and that the functional MMP activity was increased in inflamed IBD mucosa. This suggests that colonic epithelial cells, like myofibroblasts and immune cells, may contribute to local intestinal mucosal damage, through secretion of active MMPs. Disturbance of recognition and discrimination of potentially harmful pathogens from commensals in the intestinal mucosa have increasingly been implicated in the pathogenesis of IBD. Our results revealed that colonic epithelial cells express TLR9, a key pattern recognition receptor. Interestingly, the differentiated epithelial cells, which have been exposed to the luminal bacterial flora in vivo, were unresponsive to TLR9 ligand stimulation, contrasting findings in the epithelial cell line HT-29 that is cultured continuously in bacteria free environment. These findings suggest, theoretically, that colonic epithelium may regulate immune responses to microbial antigens including commensal bacterial DNA through modulation of the TLR9 pathway. Currently, the results are in line with the emerging view, that the epithelium represents an important frontline cellular component of the innate immune system in the gut. PPARγ is a nuclear receptor involved in the regulation of lipid and carbonhydrate metabolism. Recent studies in rodent colitis models suggest that PPARγ also is involved in modulation of inflammatory processes in the colon. Using the model, we characterise expression and activity of PPARs in human colonic epithelium and, additionally, evaluated the functional significance of a possible imbalanced PPARγ regulation in relation to inflammation. Our experiments showed that colonic epithelial cells express PPARγ and furthermore that PPARγ signalling was impaired in inflamed UC epithelium. It was possible to restore PPARγ signalling in the cell cultures by stimulation with rosiglitazone (a synthetic PPARγ ligand) in vitro. Hence, these experiments prompted us to design a small controlled, clinical study exploring the possible stimulatory effects of rosiglitazone (a PPAR ligand) in vivo. Interestingly, it was found that topical application of rosiglitazone in patients with active distal UC reduced clinical activity and mucosal inflammation similar to the effects measured in patients treated with mesalazine enemas. Moreover, rectal application of rosiglitazone induced PPARγ signalling in the epithelium in vivo, supporting the view that activation of PPARγ may be a new potential therapeutic target in the treatment of UC. Overall, the in vitro model of representative human colonic epithelial cells has shown to be a useful technique for detailed studies of metabolic and immunological functions that are important for homeostasis of the colonic epithelium. Currently, the findings support the view that intestinal epithelial cells actively participate in immunological processes in the colonic mucosa. Additionally, the model seems to be applicable for generating and evaluating new therapeutic approaches from laboratory bench to bed line as illustrated by the PPARγ study. It is therefore probable, that studies in models of representative colonic epithelial cells, as the one described here, could contribute with important knowledge about the pathogenesis of human inflammatory colonic diseases also in the future.

Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells.

PubMed

Meadows, Adam L; Kong, Becky; Berdichevsky, Marina; Roy, Siddhartha; Rosiva, Rosiva; Blanch, Harvey W; Clark, Douglas S

2008-01-01

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.

Cytokeratins in normal and malignant transitional epithelium. Maintenance of expression of urothelial differentiation features in transitional cell carcinomas and bladder carcinoma cell culture lines.

PubMed Central

Moll, R.; Achtstätter, T.; Becht, E.; Balcarova-Ständer, J.; Ittensohn, M.; Franke, W. W.

1988-01-01

The pattern of cytokeratins expressed in normal urothelium has been compared with that of various forms of transitional cell carcinomas (TCCs; 21 cases) and cultured bladder carcinoma cell lines, using immunolocalization and gel electrophoretic techniques. In normal urothelium, all simple-epithelium-type cytokeratins (polypeptides 7, 8, 18, 19) were detected in all cell layers, whereas antibodies to cytokeratins typical for stratified epithelia reacted with certain basal cells only or, in the case of cytokeratin 13, with cells of the basal and intermediate layers. This pattern was essentially maintained in low-grade (G1, G1/2) TCCs but was remarkably modified in G2 TCCs. In G3 TCCs simple-epithelial cytokeratins were predominant whereas the amounts of component 13 were greatly reduced. Squamous metaplasia was accompanied generally by increased or new expression of some stratified-epithelial cytokeratins. The cytokeratin patterns of cell culture lines RT-112 and RT-4 resembled those of G1 and G2 TCCs, whereas cell line T-24 was comparable to G3 carcinomas. The cell line EJ showed a markedly different pattern. The results indicate that, in the cell layers of the urothelium, the synthesis of stratification-related cytokeratins such as component 13 is inversely oriented compared with that in other stratified epithelia where these proteins are suprabasally expressed, that TCCs retain certain intrinsic cytoskeletal features of urothelium, and that different TCCs can be distinguished by their cytokeratin patterns. The potential value of these observations in histopathologic and cytologic diagnoses is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:2456018

Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar-Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Sakamoto, Atsushi; Matsumaru, Takehisa; Yamamura, Norio; Suzuki, Shinobu; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

2015-09-01

Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography-tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Force dependence of phagosome trafficking in retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

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

2014-09-01

Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

Generation of a pancreatic cancer model using a Pdx1-Flp recombinase knock-in allele

PubMed Central

Wu, Jinghai; Liu, Xin; Nayak, Sunayana G.; Pitarresi, Jason R.; Cuitiño, Maria C.; Yu, Lianbo; Hildreth, Blake E.; Thies, Katie A.; Schilling, Daniel J.; Fernandez, Soledad A.; Leone, Gustavo

2017-01-01

The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new Pdx1FlpO knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the Frt-STOP-Frt KrasG12D and p53Frt mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent in vivo tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment. PMID:28934293

Exposure of differentiated airway epithelial cells to volatile smoke in vitro.

PubMed

Beisswenger, Christoph; Platz, Juliane; Seifart, Carola; Vogelmeier, Claus; Bals, Robert

2004-01-01

Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-kappaB were applied. Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-kappaB. We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions. Copyright 2004 S. Karger AG, Basel

In vivo imaging of epithelial wound healing in the cnidarian Clytia hemisphaerica demonstrates early evolution of purse string and cell crawling closure mechanisms.

PubMed

Kamran, Zach; Zellner, Katie; Kyriazes, Harry; Kraus, Christine M; Reynier, Jean-Baptiste; Malamy, Jocelyn E

2017-12-19

All animals have mechanisms for healing damage to the epithelial sheets that cover the body and line internal cavities. Epithelial wounds heal either by cells crawling over the wound gap, by contraction of a super-cellular actin cable ("purse string") that surrounds the wound, or some combination of the two mechanisms. Both cell crawling and purse string closure of epithelial wounds are widely observed across vertebrates and invertebrates, suggesting early evolution of these mechanisms. Cnidarians evolved ~600 million years ago and are considered a sister group to the Bilateria. They have been much studied for their tremendous regenerative potential, but epithelial wound healing has not been characterized in detail. Conserved elements of wound healing in bilaterians and cnidarians would suggest an evolutionary origin in a common ancestor. Here we test this idea by characterizing epithelial wound healing in live medusae of Clytia hemisphaerica. We identified cell crawling and purse string-mediated mechanisms of healing in Clytia epithelium that appear highly analogous of those seen in higher animals, suggesting that these mechanisms may have emerged in a common ancestor. Interestingly, we found that epithelial wound healing in Clytia is 75 to >600 times faster than in cultured cells or embryos of other animals previously studied, suggesting that Clytia may provide valuable clues about optimized healing efficiency. Finally, in Clytia, we show that damage to the basement membrane in a wound gap causes a rapid shift between the cell crawling and purse string mechanisms for wound closure. This is consistent with work in other systems showing that cells marginal to a wound choose between a super-cellular actin cable or lamellipodia formation to close wounds, and suggests a mechanism underlying this decision. 1. Cell crawling and purse string mechanisms of epithelial wound healing likely evolved before the divergence of Cnidaria from the bilaterian lineage ~ 600mya 2. In Clytia, the choice between cell crawling and purse string mechanisms of wound healing depends on interactions between the epithelial cells and the basement membrane.

Inhibition of epithelial ovarian cancer by Minnelide, a water-soluble pro-drug.

PubMed

Rivard, Colleen; Geller, Melissa; Schnettler, Erica; Saluja, Manju; Vogel, Rachel Isaksson; Saluja, Ashok; Ramakrishnan, Sundaram

2014-11-01

Minnelide is a water-soluble pro-drug of triptolide, a natural product. The goal of this study was to evaluate the effectiveness of Minnelide on ovarian cancer growth in vitro and in vivo. The effect of Minnelide on ovarian cancer cell proliferation was determined by real time electrical impedance measurements. Multiple mouse models with C200 and A2780 epithelial ovarian cancer cell lines were used to assess the efficacy of Minnelide in inhibiting ovarian cancer growth. Minnelide decreased cell viability of both platinum sensitive and resistant epithelial ovarian cancer cells in vitro. Minnelide with carboplatin showed additive effects in vitro. Minnelide monotherapy increased the survival of mice bearing established ovarian tumors. Minnelide, in combination with carboplatin and paclitaxel, improved overall survival of mice. Minnelide is a promising pro-drug for the treatment of ovarian cancer, especially when combined with standard chemotherapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Epithelial-Stromal Interaction 1 (EPSTI1) Substitutes for Peritumoral Fibroblasts in the Tumor Microenvironment

PubMed Central

de Neergaard, Michala; Kim, Jiyoung; Villadsen, René; Fridriksdottir, Agla J.; Rank, Fritz; Timmermans-Wielenga, Vera; Langerød, Anita; Børresen-Dale, Anne-Lise; Petersen, Ole W.; Rønnov-Jessen, Lone

2010-01-01

Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general, no EPSTI1 staining was observed in normal breast epithelial cells from reduction mammoplasties (n = 25). However, in carcinomas, staining was positive in 22 of 40 biopsies and inversely correlated with the level of differentiation. To address the function of EPSTI1, we expressed EPSTI1 ectopically in one cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation—a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor cells, can replace peritumoral activated fibroblasts in a tumor environment assay. These observations implicate EPSTI1 as a hitherto unappreciated regulator of tumor cell properties. PMID:20133812

Nestin suppression attenuates invasive potential of endometrial cancer cells by downregulating TGF-β signaling pathway.

PubMed

Bokhari, Amber A; Baker, Tabari M; Dorjbal, Batsukh; Waheed, Sana; Zahn, Christopher M; Hamilton, Chad A; Maxwell, G Larry; Syed, Viqar

2016-10-25

Nestin, an intermediate filament protein and a stem cell marker is expressed in several tumors. Until recently, little was known about the expression levels and the role of Nestin in endometrial cancer. Compared to the immortalized endometrial epithelial cell line EM-E6/E7-TERT, endometrial cancer cell lines express high to moderate levels of Nestin. Furthermore, endometrial tumors and tumor cell lines have a cancer stem-like cell subpopulation expressing CD133. Among the cancer lines, AN3CA and KLE cells exhibited both a significantly higher number of CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE increased cells in G0/G1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G0/G1 phase and increased cells in S-phase. Nestin knockdown cells showed increased p21, p27, and PNCA levels and decreased expression of cyclin-D1 and D3. In contrast, Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by downregulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating the epithelial cell marker E-cadherin whereas the opposite was observed with Nestin overexpressing Ishikawa cells. Nestin knockdown also inhibited, while overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment.

Nestin suppression attenuates invasive potential of endometrial cancer cells by downregulating TGF-β signaling pathway

PubMed Central

Bokhari, Amber A.; Baker, Tabari M.; Dorjbal, Batsukh; Waheed, Sana; Zahn, Christopher M.; Hamilton, Chad A.; Maxwell, G. Larry; Syed, Viqar

2016-01-01

Nestin, an intermediate filament protein and a stem cell marker is expressed in several tumors. Until recently, little was known about the expression levels and the role of Nestin in endometrial cancer. Compared to the immortalized endometrial epithelial cell line EM-E6/E7-TERT, endometrial cancer cell lines express high to moderate levels of Nestin. Furthermore, endometrial tumors and tumor cell lines have a cancer stem-like cell subpopulation expressing CD133. Among the cancer lines, AN3CA and KLE cells exhibited both a significantly higher number of CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE increased cells in G0/G1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G0/G1 phase and increased cells in S-phase. Nestin knockdown cells showed increased p21, p27, and PNCA levels and decreased expression of cyclin-D1 and D3. In contrast, Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by downregulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating the epithelial cell marker E-cadherin whereas the opposite was observed with Nestin overexpressing Ishikawa cells. Nestin knockdown also inhibited, while overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment. PMID:27626172

Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

PubMed Central

Hao, Weidong; Bernard, Katie; Patel, Nita; Ulbrandt, Nancy; Feng, Hui; Svabek, Catherine; Wilson, Susan; Stracener, Christina; Wang, Kathy; Suzich, JoAnn; Blair, Wade

2012-01-01

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors. PMID:23035218

Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

PubMed Central

Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

2013-01-01

Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

Spontaneous rat nephroblastoma: ultrastructure of a transplant line.

PubMed

Hard, G C; Noble, R L

1982-08-01

Tumor tissue derived from a nephroblastoma arising spontaneously in an Nb hooded rat and carried for 56 generations as a subcutaneous syngeneic transplant was examined by electron microscopy. Histologically, the transplant showed the typical pattern of dense clusters or sheets of basophilic epithelioid cells set within ramifying tracts of fibrous mesenchyme. At the ultrastructural level, the tumor cells within dense clusters were arranged in anastomotic cords that circumscribed clear intervening spaces. The tumor cells were a monomorphic population possessing the morphologic characteristics of blast cells, including polyribosomes as the predominant cytoplasmic organelle. Furthermore, the close contiguity of cells within the cords, their polyhedral form, and their connection by intercellular junctions established the tumor cell type as epithelial. Consistent with benign stroma, mesenchymal tracts contained highly differentiated, mature fibroblasts, collagen, phagocytes, cells of the lymphoid series, and normal blood vessels. No transitional forms between epithelium and mesenchyme suggestive of bipotential differentiation were seen. Although the data represent only a single transplantation line, the observations support the contention (based on light microscopic appraisal of a number of primary tumors) that nephroblastoma in te rat can be a purely epithelial neoplasm.

Expression profile of undifferentiated cell transcription factor 1 in normal and cancerous human epithelia.

PubMed

Mouallif, Mustapha; Albert, Adelin; Zeddou, Mustapha; Ennaji, My Mustapha; Delvenne, Philippe; Guenin, Samuel

2014-08-01

Undifferentiated cell Transcription Factor 1 (UTF1) is a chromatin-bound protein involved in stem cell differentiation. It was initially reported to be restricted to stem cells or germinal tissues. However, recent work suggests that UTF1 is also expressed in somatic cells and that its expression may increase during carcinogenesis. To further clarify the expression profile of UTF1, we evaluated UTF1 expression levels immunohistochemically in eight normal human epithelia (from breast, prostate, endometrium, bladder, colon, oesophagus, lung and kidney) and their corresponding tumours as well as in several epithelial cell lines. We showed UTF1 staining in normal and tumour epithelial tissues, but with varying intensities according to the tissue location. In vitro analyses also revealed that UTF1 is expressed in somatic epithelial cell lines even in the absence of Oct4A and Sox2, its two main known regulators. The comparison of UTF1 levels in normal and tumoral tissues revealed significant overexpression in endometrial and prostatic adenocarcinomas, whereas lower intensity of the staining was observed in renal and colic tumours, suggesting a potential tissue-specific function of UTF1. Altogether, these results highlight a potential dual role for UTF1, acting either as an oncogene or as a tumour suppressor depending on the tissue. These findings also question its role as a specific marker for stem cells. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry.

PubMed

Young, Travis W; Mei, Fang C; Yang, Gong; Thompson-Lanza, Jennifer A; Liu, Jinsong; Cheng, Xiaodong

2004-07-01

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic ras(V12) allele. Of approximately 2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

Micropatterns of Matrigel for three-dimensional epithelial cultures.

PubMed

Sodunke, Temitope R; Turner, Keneshia K; Caldwell, Sarah A; McBride, Kevin W; Reginato, Mauricio J; Noh, Hongseok Moses

2007-09-01

Three-dimensional (3D) epithelial culture models are widely used to promote a physiologically relevant microenvironment for the study of normal and aberrant epithelial organization. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hindered by the lack of existing platforms for large-scale 3D cellular studies. Current 3D standard culture does not allow for single spheroid or 'acinus' analysis required for high-throughput systems. Here, we present general strategies for creating bulk micropatterns of Matrigel that can be used as a platform for 3D epithelial culture and cell-based assays at the single acinus level. Both buried and free-standing micropatterns of Matrigel were created using modified soft lithography techniques such as microtransfer molding (microTM) and dry lift-off technique. Surface modification of poly(dimethylsiloxane) (PDMS) with oxygen plasma followed by treatment with poly(2-hydroxy-ethylmethacrylate) (poly-HEMA) was sufficient to promote deformation-free release of Matrigel patterns. In addition, a novel dual-layer dry lift-off technique was developed to simultaneously generate patterns of Matrigel and poly-HEMA on a single substrate. We also demonstrate that the micropatterned Matrigel can support 3D culture originating from a single normal human mammary epithelial (MCF-10A) cell or a human breast cancer cell (MDA-MB-231) with comparable phenotypes to standard 3D culture techniques. Culture of normal MCF-10A cells on micropatterned Matrigel resulted in formation of structures with the characteristic apoptosis of centrally located cells and formation of hollow lumens. Moreover, the carcinoma cell line showed their characteristic formation of disorganized invasive cellular clusters, lacking the normal epithelial architecture on micropatterned Matrigel. Hence, micropatterned Matrigel can be used as a 3D epithelial cell-based platform for a wide variety of applications in epithelial and cancer biology, tissue engineering, as well as gene/drug screening technology.

Analysing intracellular deformation of polymer capsules using structured illumination microscopy

NASA Astrophysics Data System (ADS)

Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

2016-06-01

Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6nr02151d

Intratumoral bidirectional transitions between epithelial and mesenchymal cells in triple-negative breast cancer.

PubMed

Yamamoto, Mizuki; Sakane, Kota; Tominaga, Kana; Gotoh, Noriko; Niwa, Takayoshi; Kikuchi, Yasuko; Tada, Keiichiro; Goshima, Naoki; Semba, Kentaro; Inoue, Jun-Ichiro

2017-06-01

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition MET, are crucial in several stages of cancer metastasis. Epithelial-mesenchymal transition allows cancer cells to move to proximal blood vessels for intravasation. However, because EMT and MET processes are dynamic, mesenchymal cancer cells are likely to undergo MET transiently and subsequently re-undergo EMT to restart the metastatic process. Therefore, spatiotemporally coordinated mutual regulation between EMT and MET could occur during metastasis. To elucidate such regulation, we chose HCC38, a human triple-negative breast cancer cell line, because HCC38 is composed of epithelial and mesenchymal populations at a fixed ratio even though mesenchymal cells proliferate significantly more slowly than epithelial cells. We purified epithelial and mesenchymal cells from Venus-labeled and unlabeled HCC38 cells and mixed them at various ratios to follow EMT and MET. Using this system, we found that the efficiency of EMT is approximately an order of magnitude higher than that of MET and that the two populations significantly enhance the transition of cells from the other population to their own. In addition, knockdown of Zinc finger E-box-binding homeobox 1 (ZEB1) or Zinc finger protein SNAI2 (SLUG) significantly suppressed EMT but promoted partial MET, indicating that ZEB1 and SLUG are crucial to EMT and MET. We also show that primary breast cancer cells underwent EMT that correlated with changes in expression profiles of genes determining EMT status and breast cancer subtype. These changes were very similar to those observed in EMT in HCC38 cells. Consequently, we propose HCC38 as a suitable model to analyze EMT-MET dynamics that could affect the development of triple-negative breast cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Effect of estradiol on the expression of angiogenic factors in epithelial ovarian cancer.

PubMed

Valladares, Macarena; Plaza-Parrochia, Francisca; Lépez, Macarena; López, Daniela; Gabler, Fernando; Gayan, Patricio; Selman, Alberto; Vega, Margarita; Romero, Carmen

2017-11-01

Ovarian cancer presents a high angiogenesis (formation of new blood vessels) regulated by pro-angiogenic factors, mainly vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). An association between endogenous levels of estrogen and increased risk of developing ovarian cancer has been reported. Estrogen action is mediated by the binding to its specific receptors (ERα and ERβ), altered ERα/ERβ ratio may constitute a marker of ovarian carcinogenesis progression. To determine the effect of estradiol through ERα on the expression of NGF and VEGF in epithelial ovarian cancer (EOC). Levels of phosphorylated estrogen receptor alpha (pERα) were evaluated in well, moderate and poorly differentiated EOC samples (EOC-I, EOC-II, EOC-III). Additionally, ovarian cancer explants were stimulated with NGF (0, 10 and 100 ng/ml) and ERα, ERβ and pERα levels were detected. Finally, human ovarian surface epithelial (HOSE) and epithelial ovarian cancer (A2780) cell lines were stimulated with estradiol, where NGF and VEGF protein levels were evaluated. In tissues, ERs were detected being pERα levels significantly increased in EOC-III samples compared with EOC-I (p<0.05). Additionally, ovarian explants treated with NGF increased pERα levels meanwhile total ERα and ERβ levels did not change. Cell lines stimulated with estradiol revealed an increase of NGF and VEGF protein levels (p<0.05). Estradiol has a positive effect on pro-angiogenic factors such as NGF and VEGF expression in EOC, probably through the activation of ERα; generating a positive loop induced by NGF increasing pERα levels in epithelial ovarian cells.

Investigations on the nephrotoxicity and hepatotoxicity of trivalent and hexavalent chromium compounds.

PubMed

Dartsch, P C; Hildenbrand, S; Kimmel, R; Schmahl, F W

1998-09-01

In contrast to trivalent chromium (Cr(III)) compounds, hexavalent chromium ((Cr(VI)) compounds are oxidizing agents capable of directly inducing tissue damage and possessing carcinogenic, mutagenic and teratogenic potency. After oral or dermal absorption of Cr(VI), the kidney is the main target organ for chromium accumulation, which might result in acute tubular necrosis in humans. In contrast, an acute toxic effect of Cr(VI) on the liver has not yet been described. Therefore, we used two established epithelial cell lines from the kidney (Opossum kidney cells) and the liver (Hep G2 cells) to design an in vitro-assay which is able to examine acute toxic effects of chromium compounds. Cells of both cell lines were treated with various concentrations of Cr(III) and Cr(VI) ranging from 0.01 micromol/l to 1 mmol/l for 24 h. Thereafter, cell morphology, organization of the intracellular cytoskeleton, number of viable cells and mean cell volume were examined. The results show that Cr(VI), but not Cr(III), has an acute cytotoxic effect and causes a dose-dependent loss in cell viability. The effective dose that caused 50% of cell death was 5 micromol/l for kidney epithelial cells and 50 micromol/l for liver epithelial cells. This means that kidney epithelial cells are 10 times more sensitive towards Cr(VI) treatment than liver epithelial cells and this might explain the known nephrotoxicity in vivo. The loss in cell viability was accompanied by a rounding and detachment of the cells and a marked reduction of intracellular F-actin-containing stress fibers. Microtubules and intermediate-sized filaments were observed to be unaffected. Only in the case of kidney epithelial cells, a dose-dependent cell volume increase was observed after Cr(VI) treatment at concentrations up to 50 micromol/l. At higher concentrations, the cell volume decreased due to the high number of cells undergoing lysis and the appearance of cellular fragments. Various chloride channel blockers with different specificities, molecular structures and inhibitory potentials were tested for their ability to prevent Cr(VI)-induced cell damage. None of the channel blockers was able to inhibit cell damage, suggesting that the uptake of Cr(VI) through the general anion transport system of the cell membrane might be only one facet of cellular uptake and toxification. The data presented here not only confirm the different organ-specific effects of Cr(III) and Cr(VI), but also provide a basis for future experiments on the understanding of acute toxicity of Cr(VI) compounds. Moreover, the results demonstrate that the designed in vitro-assay might be a useful tool to prove whether non-toxic Cr(III) can be oxidized to Cr(VI) under specific industrial conditions (for example, in the leather or chrome industry).

E-cigarette vapour is not inert and exposure can lead to cell damage.

PubMed

Holliday, Richard; Kist, Ralf; Bauld, Linda

2016-03-01

In vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. The widely available cell line HaCat, a spontaneously transformed immortal keratinocyte and the HNSCC cell lines HN30 and UMSCC10B were used. Cells were exposed to nicotine-containing and nicotine-free vapour extract from two popular e-cigarette brands for periods ranging from 48 hours to eight weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapour nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing two brands of e-cigs show that at biologically relevant doses, vapourised e-cig liquids induce increased DNA strand breaks and cell death, and decreased clono- genic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects of e-cig usage, as well as whether the DNA damage shown in our study as a result of e-cig exposure will lead to mutations that ultimately result in cancer.

Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)

PubMed Central

Rizo, Walace Fraga; Ferreira, Luis Eduardo; Colnaghi, Vanessa; Martins, Juliana Simões; Franchi, Leonardo Pereira; Takahashi, Catarina Satie; Beleboni, Rene Oliveira; Marins, Mozart; Pereira, Paulo Sérgio; Fachin, Ana Lúcia

2013-01-01

Cancer has become a major public health problem worldwide and the number of deaths due to this disease is increasing almost exponentially. In the constant search for new treatments, natural products of plant origin have provided a variety of new compounds to be explored as antitumor agents. Tabernaemontana catharinensis is a medicinal plant that produces alkaloids with expressive antitumor activity, such as heyneanine, coronaridine and voacangine. The aim of present study was firstly to screen the cytotoxic activity of the indole alkaloids heyneanine, coronaridine and voacangine against HeLa (human cervix tumor), 3T3 (normal mouse embryo fibroblasts), Hep-2 (human laryngeal epithelial carcinoma) and B-16 (murine skin) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); and secondly to analyze the apoptotic activity, cell membrane damage and genotoxicity of the compound that showed the best cytotoxic activity against the tumor cell lines tested. Coronaridine was the one that exhibited greater cytotoxic activity in the laryngeal carcinoma cell line Hep-2 (IC50 = 54.47 μg/mL) than the other alkaloids tested (voacangine IC50 = 159.33 g/mL, and heyneanine IC50 = 689.45 μg/mL). Coronaridine induced apoptosis in cell lines 3T3 and Hep-2, even at high concentrations. The evaluation of genotoxicity by comet assay showed further that coronaridine caused minimal DNA damage in the Hep-2 tumor cell line, and the LDH test showed that it did not affect the plasma membrane. These results suggest that further investigation of coronaridine as an antitumor agent has merit. PMID:23569415

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma

PubMed Central

Campo-Verde-Arbocco, Fiorella; López-Laur, José D.; Romeo, Leonardo R.; Giorlando, Noelia; Bruna, Flavia A.; Contador, David E.; López-Fontana, Gastón; Santiano, Flavia E.; Sasso, Corina V.; Zyla, Leila E.; López-Fontana, Constanza M.; Calvo, Juan C.; Carón, Rubén W.; Creydt, Virginia Pistone

2017-01-01

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease. PMID:29212223

C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

PubMed

Karnati, Srikanth; Palaniswamy, Saranya; Alam, Mohammad Rashedul; Oruqaj, Gani; Stamme, Cordula; Baumgart-Vogt, Eveline

2016-03-01

In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

Magnolol inhibits LPS-induced inflammatory response in uterine epithelial cells : magnolol inhibits LPS-induced inflammatory response.

PubMed

Luo, Jia; Xu, Yanwen; Zhang, Minfang; Gao, Ling; Fang, Cong; Zhou, Canquan

2013-10-01

Endometritis is an inflammation of the uterine lining that is commonly initiated at parturition. The uterine epithelial cells play an important role in defending against invading pathogens. Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been shown to have anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effect of magnolol in modifying lipopolysaccharide (LPS)-induced signal pathways in mouse uterine epithelial cells. We found that magnolol inhibited TNF-α and IL-6 production in LPS-stimulated mouse uterine epithelial cells. We also found that magnolol inhibited LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK, and P38. Furthermore, magnolol could significantly inhibit the expression of TLR4 stimulating by LPS. These results suggest that magnolol exerts an anti-inflammatory property by downregulating the expression of TLR4 upregulated by LPS, thereby attenuating TLR4-mediated NF-κB and MAPK signaling and the release of pro-inflammatory cytokines. These findings suggest that magnolol may be a therapeutic agent against endometritis.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

PubMed

McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

2011-04-01

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

PubMed

Irshad, Shazia; Bansal, Mukesh; Guarnieri, Paolo; Davis, Hayley; Al Haj Zen, Ayman; Baran, Brygida; Pinna, Claudia Maria Assunta; Rahman, Haseeb; Biswas, Sujata; Bardella, Chiara; Jeffery, Rosemary; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Lewis, Annabelle; Leedham, Simon John

2017-06-01

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis.

PubMed

Kudinov, Alexander E; Deneka, Alexander; Nikonova, Anna S; Beck, Tim N; Ahn, Young-Ho; Liu, Xin; Martinez, Cathleen F; Schultz, Fred A; Reynolds, Samuel; Yang, Dong-Hua; Cai, Kathy Q; Yaghmour, Khaled M; Baker, Karmel A; Egleston, Brian L; Nicolas, Emmanuelle; Chikwem, Adaeze; Andrianov, Gregory; Singh, Shelly; Borghaei, Hossein; Serebriiskii, Ilya G; Gibbons, Don L; Kurie, Jonathan M; Golemis, Erica A; Boumber, Yanis

2016-06-21

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

Medroxyprogesterone acetate-treated human, primary endometrial epithelial cells reveal unique gene expression signature linked to innate immunity and HIV-1 susceptibility.

PubMed

Woods, Matthew W; Zahoor, Muhammad Atif; Dizzell, Sara; Verschoor, Chris P; Kaushic, Charu

2018-01-01

Medroxyprogesterone acetate (MPA), a progestin-based hormonal contraceptive designed to mimic progesterone, has been linked to increased human immunodeficiency virus (HIV-1) susceptibility. Genital epithelial cells (GECs) form the mucosal lining of the female genital tract (FGT) and provide the first line of protection against HIV-1. The impact of endogenous sex hormones or MPA on the gene expression profile of GECs has not been comprehensively documented. Using microarray analysis, we characterized the transcriptional profile of primary endometrial epithelial cells grown in physiological levels of E2, P4, and MPA. Each hormone treatment altered the gene expression profile of GECs in a unique manner. Interestingly, although MPA is a progestogen, the gene expression profile induced by it was distinct from P4. MPA increased gene expression of genes related to inflammation and cholesterol synthesis linked to innate immunity and HIV-1 susceptibility. The analysis of gene expression profiles provides insights into the effects of sex hormones and MPA on GECs and allows us to posit possible mechanisms of the MPA-mediated increase in HIV-1 acquisition. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Protein kinase D is increased and activated in lung epithelial cells and macrophages in idiopathic pulmonary fibrosis.

PubMed

Gan, Huachen; McKenzie, Raymond; Hao, Qin; Idell, Steven; Tang, Hua

2014-01-01

Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.

Characterization of fibroblast-free CWR-R1ca castration-recurrent prostate cancer cell line.

PubMed

Shourideh, Mojgan; DePriest, Adam; Mohler, James L; Wilson, Elizabeth M; Koochekpour, Shahriar

2016-09-01

The previously established CWR-R1 cell line has been used as an in vitro model representing castration-recurrent prostate cancer. Microscopic observation of subconfluent cells demonstrated two distinct cellular morphologies: polygonal closely aggregated epithelial cells surrounded by bipolar fibroblastic cells with long processes. This study sought to establish and characterize a fibroblast-free derivative of the CWR-R1 cell line. The CWR-R1ca cell line was established from CWR-R1 cells by removing fibroblasts using multiple cycles of short-term trypsinization, cloning, and pooling single-cell colonies. Authentication of fibroblast-free CWR-R1ca cells was demonstrated by analyzing the expression of cytodifferentiation and prostate-associated markers, DNA and cytogenetic profiling, and growth pattern in the absence or presence of androgen. CWR-R1ca is an androgen-sensitive cell line that expresses the androgen receptor (AR) and its splice variant 7 and the luminal epithelia markers, CK-8, CK-18, and c-Met. CWR-R1fb fibroblasts isolated from CWR-R1 cells express AR, hepatocyte growth factor-α, and mouse β-actin but not AR-V7 or epithelial markers. Cytogenetic analysis of CWR-R1ca cells revealed a hyperdiploid male with numerical gains in chromosomes 1, 7, 8, 10, 11, and 12, deletion of one chromosome 2 allele, structural abnormalities that include der(1)t(1:4), der(4)t(2:4), der(10)t(4:10), and an unbalanced reciprocal translocation between chromosome 6 and 14. DNA-profiling revealed that CWR-R1ca cells had significant short-tandem repeat marker homology with CWR22Pc and CWR22Rv1 cell lines, which indicated lineage derivation from CWR22 prostate cancer xenografts. CWR-R1ca cells were responsive to the growth stimulatory effects of dihydrotestosterone (DHT) in the femtomolar range. This study establishes CWR-R1ca cells as a fibroblast-free derivative of the castration-recurrent CWR-R1 cell line. Prostate 76:1067-1077, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells.

PubMed

Wen, G; Pachner, L I; Gessner, D K; Eder, K; Ringseis, R

2016-11-01

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the mutated SRE at -38 of the NIS 5'-flanking region showed that in vitro-translated nSREBP-1c and nSREBP-2 bind only the wild-type but not the mutated SRE at -38 of NIS. Collectively, the present results from cell culture experiments with human mammary epithelial MCF-7 cells and from genetic studies show for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells. Future studies are necessary to clarify if the regulation of NIS expression and iodide uptake by SREBP also applies to the lactating bovine mammary epithelium. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

EMP-1 is a junctional protein in a liver stem cell line and in the liver.

PubMed

Lee, Hsuan-Shu; Sherley, James L; Chen, Jeremy J W; Chiu, Chien-Chang; Chiou, Ling-Ling; Liang, Ja-Der; Yang, Pan-Chyr; Huang, Guan-Tarn; Sheu, Jin-Chuan

2005-09-09

In an attempt to discover cell markers for liver stem cells, a cDNA microarray analysis was carried out to compare the gene expression profiles between an adult liver stem cell line, Lig-8, and mature hepatocytes. Several genes in the categories of extracellular matrix, cell membrane, cell adhesion, transcription factor, signal molecule, transporter, and metabolic enzyme were shown to be differentially expressed in Lig-8 cells. Among them, epithelial membrane protein (EMP)-1 has been previously implicated with stem cell phenotypes. Antiserum to EMP-1 was produced to localize its expression. On monolayers of Lig-8 cells, EMP-1 was expressed along the intercellular border. In the liver harboring proliferating oval cells, the liver progenitors, EMP-1 was localized as ribbon bands, a staining pattern for epithelial junctions, all the way through bile duct epithelia, oval cell ductules, and into peri-hepatocytic regions. These peri-hepatocytic regions were proved to be bile canaliculi by co-localization of EMP-1 and dipeptidyl peptidase IV, an enzyme located on bile canaliculi. This report is the first to indicate EMP-1 to be a junctional protein in the liver.

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

PubMed

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

2011-01-01

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

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

PubMed Central

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

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44high/CD24low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer. PMID:21915264

Epithelial morphogenesis of germline-derived pluripotent stem cells on organotypic skin equivalents in vitro.

PubMed

van de Kamp, Julia; Kramann, Rafael; Anraths, Julia; Schöler, Hans R; Ko, Kinarm; Knüchel, Ruth; Zenke, Martin; Neuss, Sabine; Schneider, Rebekka K

2012-03-01

For tissue engineering, cultivation of pluripotent stem cells on three-dimensional scaffolds allows the generation of organ-like structures. Previously, we have established an organotypic culture system of skin to induce epidermal differentiation in adult stem cells. Multipotent stem cells are not able to differentiate across germinal boundaries. In contrast, pluripotent stem cells readily differentiate into tissues of all three germ layers. Germline-derived pluripotent stem cells (gPS cells) can be generated by induction of pluripotency in mouse unipotent germline stem cells without the introduction of exogenous transcription factors. In the current study, we analyzed the influence of organotypic culture conditions of skin on the epithelial differentiation of gPS cells in comparison to the well-established HM1 ES cell line. Quantitative RT-PCR data of the pluripotency gene Oct4 showed that gPS cells are characterized by an accelerated Oct4-downregulation compared to HM1 ES cells. When subjected to the organotypic culture conditions of skin, gPS cells formed tubulocystic structures lined by stratified (CK5/6(+), CK14(+), CK8/18(-)) epithelia. HM1 ES cells formed only small tubulocystic structures lined by simple, CK8/18(+) epithelia. BMP-4, an epidermal morphogen, significantly enhanced the expression of epithelial markers in HM1 ES cells, but did not significantly affect the formation of complex (squamous) epithelia in gPS cells. In HM1 ES cells the differentiation into squamous epithelium was only inducible in the presence of mature dermal fibroblasts. Both pluripotent stem cell types spontaneously differentiated into mesodermal, endodermal and into neuroectodermal cells at low frequency, underlining their pluripotent differentiation capacity. Concluding, the organotypic culture conditions of skin induce a multilayered, stratified epithelium in gPS cells, in HM1 ES cells only in the presence of dermal fibroblasts. Thus, our data show that differentiation protocols strongly depend on the stem cell type and have to be modified for each specific stem cell type. Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Upregulation of CD147 Promotes Metastasis of Cholangiocarcinoma by Modulating the Epithelial-to-Mesenchymal Transitional Process.

PubMed

Dana, Paweena; Kariya, Ryusho; Vaeteewoottacharn, Kulthida; Sawanyawisuth, Kanlayanee; Seubwai, Wunchana; Matsuda, Kouki; Okada, Seiji; Wongkham, Sopit

2017-08-07

CD147 is a transmembrane protein that can induce the expression and activity of matrix metalloproteinases (MMPs). Expression of CD147 has been shown to potentiate cell migration, invasion, and metastasis of cancer. In this study, the critical role of CD147 in metastasis was elucidated using CD147-overexpressing cholangiocarcinoma (CCA) cells in vitro and in vivo. The molecular mechanism, demonstrated herein, supported the hypothesis that metastasis increased in CD147-overexpressing cells. Five CD147-overexpressing clones (Ex-CD147) were established from a low CD147-expressing CCA cell line, KKU-055, using lentivirus containing pReceiver-Lenti-CD147. The metastatic capability was determined using the tail vein injection mouse model and an in vitro 3D invasion assay. Liver colonization was assessed using anti-HLA class I immunohistochemistry. Adhesion abilities, cytoskeletal arrangements, MMP activities, the expressions of adhesion molecules, and epithelial-mesenchymal transitional markers were analyzed. All Ex-CD147 clones exhibited a high CD147 expression and high liver colonization in the tail vein-injected mouse model, whereas parental cells lacked this ability. Ex-CD147 clones exhibited metastatic phenotypes (i.e., an increase in F-actin rearrangement) and cell invasion and a decrease in cell adhesion. The molecular mechanisms were shown to be via the induction of MMP-2 activity and enhancement of epithelial-mesenchymal transitions. An increase in mesenchymal markers Slug, vimentin, and N-cadherin, and a decrease in epithelial markers E-cadherin and claudin-1, together with suppression of the adhesion molecule ICAM-1, were observed in the Ex-CD147 clones. Moreover, suppression of CD147 expression using siCD147 in two CCA cell lines with high CD147 expression significantly decreased cell migration and invasion of these CCA cells. These findings emphasize the essential role of CD147 in CCA metastasis and suggest CD147 as a promising target for the effective treatment of CCA.

Sulforaphane-stimulated phase II enzyme induction inhibits cytokine production by airway epithelial cells stimulated with diesel extract.

PubMed

Ritz, Stacey A; Wan, Junxiang; Diaz-Sanchez, David

2007-01-01

Airborne particulate pollutants, such as diesel exhaust particles, are thought to exacerbate lung and cardiovascular diseases through induction of oxidative stress. Sulforaphane, derived from cruciferous vegetables, is the most potent known inducer of phase II enzymes involved in the detoxification of xenobiotics. We postulated that sulforaphane may be able to ameliorate the adverse effects of pollutants by upregulating expression of endogenous antioxidant enzymes. Stimulation of bronchial epithelial cells with the chemical constituents of diesel particles result in the production of proinflammatory cytokines. We first demonstrated a role for phase II enzymes in regulating diesel effects by transfecting the airway epithelial cell line (BEAS-2B) with the sentinel phase II enzyme NAD(P)H: quinine oxidoreductase 1 (NQO1). IL-8 production in response to diesel extract was significantly reduced in these compared with untransfected cells. We then examined whether sulforaphane would stimulate phase II induction and whether this would thereby ablate the effect of diesel extracts on cytokine production. We verified that sulforaphane significantly augmented expression of the phase II enzyme genes GSTM1 and NQO1 and confirmed that sulforaphane treatment increased glutathione S-transferase activity in epithelial cells without inducing cell death or apoptosis. Sulforaphane pretreatment inhibited IL-8 production by BEAS-2B cells upon stimulation with diesel extract. Similarly, whereas diesel extract stimulated production of IL-8, granulocyte-macrophage colony-stimulating factor, and IL-1beta from primary human bronchial epithelial cells, sulforaphane pretreatment inhibited diesel-induced production of all of these cytokines. Our studies show that sulforaphane can mitigate the effect of diesel in respiratory epithelial cells and demonstrate the chemopreventative potential of phase II enzyme enhancement.

Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

PubMed Central

Yan, Bo-jing; Wu, Zhi-zhong; Chong, Wei-hua; Li, Gen-lin

2016-01-01

Several studies have investigated the protective functions of brain-derived neurotrophic factor (BDNF) in retinitis pigmentosa. However, a BDNF-based therapy for retinitis pigmentosa is not yet available. To develop an efficient treatment for fundus disease, an eukaryotic expression plasmid was generated and used to transfect human 293T cells to assess the expression and bioactivity of BDNF on acute retinal pigment epithelial-19 (ARPE-19) cells, a human retinal epithelial cell line. After 96 hours of co-culture in a Transwell chamber, ARPE-19 cells exposed to BDNF secreted by 293T cells were more viable than ARPE-19 cells not exposed to secreted BDNF. Western blot assay showed that Bax levels were downregulated and that Bcl-2 levels were upregulated in human ARPE-19 cells exposed to BDNF. Furthermore, 293T cells transfected with the BDNF gene steadily secreted the protein. The powerful anti-apoptotic function of this BDNF may be useful for the treatment of retinitis pigmentosa and other retinal degenerative diseases. PMID:28197196

The In Vitro Differentiation of GDNF Gene-Engineered Amniotic Fluid-Derived Stem Cells into Renal Tubular Epithelial-Like Cells.

PubMed

Lu, Ying; Wang, Zhuojun; Chen, Lu; Wang, Jia; Li, Shulin; Liu, Caixia; Sun, Dong

2018-05-01

Amniotic fluid is an alternative source of stem cells, and human amniotic fluid-derived stem cells (AFSCs) obtained from a small amount of amniotic fluid collected during the second trimester represent a novel source for use in regenerative medicine. These AFSCs are characterized by lower diversity, a higher proliferation rate, and a wider differentiation capability than adult mesenchymal stem cells. AFSCs are selected based on the cell surface marker c-kit receptor (CD117) using immunomagnetic sorting. Glial cell line-derived neurotrophic factor (GDNF) is expressed during early kidney development and regulates the proliferation and differentiation of stem cells in vitro. In this study, c-kit-sorted AFSCs were induced toward osteogenic or adipogenic differentiation. AFSCs engineered via the insertion of GDNF were cocultured with mouse renal tubular epithelial cells (mRTECs), which were preconditioned by hypoxia-reoxygenation in vitro. After coculture for 8 days, AFSCs differentiation into epithelial-like cells was evaluated by performing immunofluorescence, flow cytometry, and quantitative real-time polymerase chain reaction to identify cells expressing the renal epithelial markers, cytokeratin 18 (CK18), E-cadherin, aquaporin-1 (AQP1), and paired box 2 gene (Pax2). The GDNF gene enhanced AFSCs differentiation into RTECs. AFSCs possess self-renewal ability and multiple differentiation potential and thus represent a new source of stem cells.

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

PubMed Central

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

2015-01-01

Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/mammalian target of rapamycin and p38 MAPK mediated pathways. PMID:25632222

Synthesis and characterization of 2-substituted benzimidazoles and their evaluation as anticancer agent

NASA Astrophysics Data System (ADS)

Azam, Mohammad; Khan, Azmat Ali; Al-Resayes, Saud I.; Islam, Mohammad Shahidul; Saxena, Ajit Kumar; Dwivedi, Sourabh; Musarrat, Javed; Trzesowska-Kruszynska, Agata; Kruszynski, Rafal

2015-05-01

In this work, we report a series of benzimidazole derivatives synthesized from benzene-1,2-diamine and aryl-aldehydes at room temperature. The synthesized compounds have been characterized on the basis of elemental analysis and various spectroscopic studies viz., IR, 1H- and 13C-NMR, ESI-MS as well by X-ray single X-ray crystallographic study. Interaction of these compounds with CT-DNA has been examined with fluorescence experiments and showed significant binding ability. All the synthesized compounds have been screened for their antitumor activities against various human cancer cell lines viz., Human breast adenocarcinoma cell line (MCF-7), Human leukemia cell line (THP-1), Human prostate cancer cell lines (PC-3) and adenocarcinomic human alveolar basal epithelial cell lines (A-549). Interestingly, all the compounds showed significant anticancer activity.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

PubMed

Liu, Y; Egyhazi, S; Hansson, J; Bhide, S V; Kulkarni, P S; Grafström, R C

1997-10-01

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

PubMed

Wang, Kening; Kappel, Justin D; Canders, Caleb; Davila, Wilmer F; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I

2012-12-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

A Herpes Simplex Virus 2 Glycoprotein D Mutant Generated by Bacterial Artificial Chromosome Mutagenesis Is Severely Impaired for Infecting Neuronal Cells and Infects Only Vero Cells Expressing Exogenous HVEM

PubMed Central

Kappel, Justin D.; Canders, Caleb; Davila, Wilmer F.; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I.

2012-01-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine. PMID:22993162

Adaptations to the air breathing in the posterior intestine of the catfish (Corydoras aeneus, Callichthyidae). A histological and ultrastructural study.

PubMed

Podkowa, Dagmara; Goniakowska-Witalińska, Lucyna

2002-01-01

A light and transmission electron microscopic study of the intestine of catfish C. aeneus shows that the anterior part of the intestine is a site of digestion and absorption and its structure is typical of that of other teleostean fishes. However, in this species the thin-walled posterior intestine is adapted to air breathing. In this region mucosa is smooth and lined with respiratory epithelium with capillary network. Several types of cells are observed in the epithelium: flattened respiratory epithelial cells with short microvili, goblet cells, scarce epithelial cells with numerous longer microvilli, and two types of endocrine cells (EC). The solitary brush cells with several long and thick microvilli described here are the first observation of such cells in the gastrointestinal tract of fishes. Bodies of respiratory epithelial cells lie between capillaries. Their cytoplasm, apart from typical organelles contains dense and lamellar bodies, which are a site of accumulation of surfactant. In regions where capillaries are covered by thin cytoplasmic sheets of respiratory epithelial cells, a thin (0.24-3.00 microm) air-blood barrier is formed, thus enabling gas exchange. Epithelial cells with longer microvilli do not participate in the formation of the air-blood barrier and are probably responsible for absorbtion. EC of the closed type are dispersed within the epithelium. Their cytoplasm contains characteristic round or oval dense core vesicles 69 to 230 nm in diameter. The role of EC and brush cells in the regulation of processes related to absorbtion, and to respiration, is disscused.

Morphology of the ampullae of Lorenzini in juvenile freshwater Carcharhinus leucas.

PubMed

Whitehead, Darryl L; Gauthier, Arnault R G; Mu, Erica W H; Bennett, Mike B; Tibbetts, Ian R

2015-05-01

Ampullae of Lorenzini were examined from juvenile Carcharhinus leucas (831-1,045 mm total length) captured from freshwater regions of the Brisbane River. The ampullary organ structure differs from all other previously described ampullae in the canal wall structure, the general shape of the ampullary canal, and the apically nucleated supportive cells. Ampullary pores of 140-205 µm in diameter are distributed over the surface of the head region with 2,681 and 2,913 pores present in two sharks that were studied in detail. The primary variation of the ampullary organs appears in the canal epithelial cells which occur as either flattened squamous epithelial cells or a second form of pseudostratified contour-ridged epithelial cells; both cell types appear to release material into the ampullary lumen. Secondarily, this ampullary canal varies due to involuted walls that form a clover-like canal wall structure. At the proximal end of the canal, contour-ridged cells abut a narrow region of cuboidal epithelial cells that verge on the constant, six alveolar sacs of the ampulla. The alveolar sacs contain numerous receptor and supportive cells bound by tight junctions and desmosomes. Pear-shaped receptor cells that possess a single apical kinocilium are connected basally by unmyelinated neural boutons. Opposed to previously described ampullae of Lorenzini, the supportive cells have an apical nucleus, possess a low number of microvilli, and form a unique, jagged alveolar wall. A centrally positioned centrum cap of cuboidal epithelial cells overlies a primary afferent lateral line nerve. © 2014 Wiley Periodicals, Inc.

Structural changes in endometrial basal glands during menstruation.

PubMed

Garry, R; Hart, R; Karthigasu, K A; Burke, C

2010-09-01

To prospectively observe the changes occurring in endometrial glandular morphology during menstrual shedding and regeneration. Prospective observational study. The academic gynaecological endoscopy unit of a university teaching hospital. Population Thirteen patients investigated for a variety of benign, non-infective gynaecological disorders during the active bleeding phase of the menstrual cycle. The morphological appearances of concurrent histological and scanning electron microscopic images of endometrium taken at different stages of the active bleeding phase of menstruation were studied and correlated with the simultaneous immunohistochemical expression of the Ki-67 proliferation marker and the CD68 marker of macrophage activity. Change in morphology of endometrial glands at various stages of menstruation. Endometrial glands within the basalis show evidence of apoptosis and associated macrophage activity immediately before and during menstruation. There is subsequent destruction and removal of old secretory glandular epithelial elements, and rapid replacement with new narrow glands lined with small epithelial cells. There is no evidence of mitotic cell division or expression of Ki-67 in the glandular cells during this replacement process, but there is evidence of marked macrophage activity prior to glandular cell loss. Early endometrial epithelial repair after menstruation is not a consequence of mitotic cell division. It occurs without evidence of Ki-67 expression. There is structural evidence of programmed cell death and intense macrophage activity associated with glandular remodelling. Dead epithelial cells are shed from the glands and accumulate within the endometrial cavity to be replaced by new small epithelial cells that appear to arise by differentiation of the surrounding stromal cells. We propose that these stromal cells are endometrial progenitor/stem cells.

The role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer.

PubMed

Belotte, Jimmy; Fletcher, Nicole M; Awonuga, Awoniyi O; Alexis, Mitchell; Abu-Soud, Husam M; Saed, Mohammed G; Diamond, Michael P; Saed, Ghassan M

2014-04-01

To investigate the role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer (EOC). Two parent EOC cell lines (MDAH-2774 and SKOV-3) and their chemoresistant counterparts (cisplatin, 50 µmol/L) were used. Total RNA was extracted and subjected to real-time reverse transcriptase polymerase chain reaction to evaluate the expression of glutathione reductase (GSR) and inducible nitric oxide synthase (iNOS), as well as nitrate/nitrite levels. Analysis of variance was used for main effects and Tukey for post hoc analysis at P < .05 for statistical significance. Both cisplatin resistant cell lines displayed a significant decrease in GSR messenger RNA (mRNA) levels and activity (P < .01). As compared to sensitive controls, nitrate/nitrite levels were significantly higher in SKOV-3 cisplatin resistant cells while iNOS mRNA levels were significantly higher in MDAH-2774 cisplatin resistant cells (P < .05). Our data suggest that the development of cisplatin resistance tilts the balance toward a pro-oxidant state in EOC.

Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460

PubMed Central

Liu, Zhi-Hua; Shen, Tong-Yi; Zhang, Peng; Ma, Yan-Lei; Moyer, Mary Pat; Qin, Huan-Long

2010-01-01

AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli) or enteroinvasive E. coli. L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells. CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli, and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases. PMID:21128328

University of Texas Southwestern Medical Center: NSCLC Cell Lines with Loss of SMARCA4 Expression are Hypersensitive to Inhibitors of Aurora Kinase A | Office of Cancer Genomics

Cancer.gov

A genome-wide siRNA screen was employed to identify genes that were selectively toxic for a non-small cell lung cancer (NSCLC) cell line that lacked expression of SMARCA4, but were not toxic in non-cancerous immortalized human bronchial epithelial cells lacking SMARCA4 expression. Among the selectively toxic genes were several mapping to the molecular machinery regulating activity of Aurora kinase A on the mitotic spindle.

University of Texas Southwestern Medical Center (UTSW): NSCLC Cell Lines with Loss of SMARCA4 Expression are Hypersensitive to Inhibitors of Aurora Kinase A | Office of Cancer Genomics

Cancer.gov

A genome-wide siRNA screen was employed to identify genes that were selectively toxic for a non-small cell lung cancer (NSCLC) cell line that lacked expression of SMARCA4, but were not toxic in non-cancerous immortalized human bronchial epithelial cells lacking SMARCA4 expression. Among the selectively toxic genes were several mapping to the molecular machinery regulating activity of Aurora kinase A on the mitotic spindle.

Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases.

PubMed

Aldhahrani, Adil; Verdon, Bernard; Ward, Chris; Pearson, Jeffery

2017-01-01

Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B). The immortalised human bronchial epithelial cell line (BEAS-2B) was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL)-8, IL-6 and granulocyte-macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L -1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L -1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B. Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo . This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury.

Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1.

PubMed Central

Soler, C; Felipe, A; Casado, F J; McGivan, J D; Pastor-Anglada, M

1993-01-01

Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms. PMID:8435065

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

PubMed

Flood, P; Alvarez, L; Reynaud, E G

2016-10-11

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

PubMed Central

Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

2013-01-01

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

IL-29 Enhances CXCL10 Production in TNF-α-stimulated Human Oral Epithelial Cells.

PubMed

Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

2017-08-01

Interleukin-29 (IL-29) is a cytokine belonging to the Type III interferon family. It was recently detected in the gingival crevicular fluid of periodontitis patients. However, the role of IL-29 in the pathogenesis of periodontal disease remains unknown. The aim of this study was to examine the effects of IL-29 on C-X-C motif chemokine ligand 10 (CXCL10) production in human oral epithelial cells. We measured CXCL10 production in TR146 cells, which is a human oral epithelial cell line, using an enzyme-linked immunosorbent assay. We used a Western blot analysis to detect IL-29 receptor expression and the phosphorylation levels of signal transduction molecules, including p38 mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor (NF)- κB p65, in the TR146 cells. The TR146 cells expressed the IL-29 receptor. IL-29 induced CXCL10 production in the TR146 cells. IL-29 significantly enhanced CXCL10 production in tumor necrosis factor (TNF)-α-stimulated TR146 cells. The p38 MAPK, STAT3, and NF-κB pathways were found to be related to the IL-29-induced enhancement of CXCL10 production in TNF-α-stimulated TR146 cells. IL-29 promotes T helper 1-cell accumulation in periodontal lesions by inducing CXCL10 production in oral epithelial cells.

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

PubMed

Jenny, Robert A; Hirst, Claire; Lim, Sue Mei; Goulburn, Adam L; Micallef, Suzanne J; Labonne, Tanya; Kicic, Anthony; Ling, Kak-Ming; Stick, Stephen M; Ng, Elizabeth S; Trounson, Alan; Giudice, Antonietta; Elefanty, Andrew G; Stanley, Edouard G

2015-06-01

Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold. ©AlphaMed Press.

[The epithelial junctional zones of the anal canal and cervix uterus: the ultrastructure of tumors of these zones].

PubMed

Chernyĭ, A P; Iakovleva, N I

1990-01-01

Relationships between squamous and columnar epithelia in the anal canal and cervix uteri of postnatal period and fetus were studied. The transitional stratified epithelial lining, which is called junctional epithelium, is interposed between the mentioned epithelia. The junctional epithelium has variable numbers of layers of epidermoid cells, which differ from cells of atypical squamous epithelium by some ultrastructural features of the cytoskeleton and cell surface and by a low content of glycogen. The hypothesis on the physiological significance of this epithelium is proposed. Ultrastructural features of the cytoskeleton and cell surface suggest that anal basaloid carcinomas and some cervical squamous carcinomas may develop from so-called junctional epithelium.

Promotion of a cancer-like phenotype, through chronic exposure to inflammatory cytokines and hypoxia in a bronchial epithelial cell line model

PubMed Central

Baird, Anne-Marie; Gray, Steven G.; Richard, Derek J.; O’Byrne, Kenneth J.

2016-01-01

Globally, lung cancer accounts for approximately 20% of all cancer related deaths. Five-year survival is poor and rates have remained unchanged for the past four decades. There is an urgent need to identify markers of lung carcinogenesis and new targets for therapy. Given the recent successes of immune modulators in cancer therapy and the improved understanding of immune evasion by tumours, we sought to determine the carcinogenic impact of chronic TNF-α and IL-1β exposure in a normal bronchial epithelial cell line model. Following three months of culture in a chronic inflammatory environment under conditions of normoxia and hypoxia (0.5% oxygen), normal cells developed a number of key genotypic and phenotypic alterations. Important cellular features such as the proliferative, adhesive and invasive capacity of the normal cells were significantly amplified. In addition, gene expression profiles were altered in pathways associated with apoptosis, angiogenesis and invasion. The data generated in this study provides support that TNF-α, IL-1β and hypoxia promotes a neoplastic phenotype in normal bronchial epithelial cells. In turn these mediators may be of benefit for biomarker and/or immune-therapy target studies. This project provides an important inflammatory in vitro model for further immuno-oncology studies in the lung cancer setting. PMID:26759080

Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

PubMed

Feng, Dingqing; Yan, Keqin; Zhou, Ying; Liang, Haiyan; Liang, Jing; Zhao, Weidong; Dong, Zhongjun; Ling, Bin

2016-10-04

The human papillomavirus (HPV) oncoproteins E6 and E7 are risk factors that are primarily responsible for the initiation and progression of cervical cancer, and they play a key role in immortalization and transformation by reprogramming differentiating host epithelial cells. It is unclear how cervical epithelial cells transform into tumor-initiating cells (TICs). Here, we observed that the germ stem cell protein Piwil2 is expressed in pre-cancerous and malignant lesions of the cervix and cervical cancer cell lines with the exception of the non-HPV-infected C33a cell line. Knockdown of Piwil2 by shRNA led to a marked reduction in proliferation and colony formation, in vivo tumorigenicity, chemo-resistance, and the proportion of cancer stem-like cells. In contrast, Piwil2 overexpression induced malignant transformation of HaCaT cells and the acquisition of tumor-initiating capabilities. Gene-set enrichment analysis revealed embryonic stem cell (ESC) identity, malignant biological behavior, and specifically, activation targets of the cell reprogramming factors c-Myc, Klf4, Nanog, Oct4, and Sox2 in Piwil2-overexpressing HaCaT cells. We further confirmed that E6 and E7 reactivated Piwil2 and that E6 and E7 overexpression resulted in a similar gene-set enrichment pattern as Piwil2 overexpression in HaCaT cells. Moreover, Piwil2 overexpression or E6 and E7 activation induced H3K9 acetylation but reduced H3K9 trimethylation, which contributed to the epigenetic reprogramming and ESC signature maintenance, as predicted previously. Our study demonstrates that Piwil2, reactivated by the HPV oncoproteins E6 and E7, plays an essential role in the transformation of cervical epithelial cells to TICs via epigenetics-based cell reprogramming.

The Neonatal Fc Receptor (FcRn) Enhances Human Immunodeficiency Virus Type 1 (HIV-1) Transcytosis across Epithelial Cells

PubMed Central

Gupta, Sandeep; Gach, Johannes S.; Becerra, Juan C.; Phan, Tran B.; Pudney, Jeffrey; Moldoveanu, Zina; Joseph, Sarah B.; Landucci, Gary; Supnet, Medalyn Jude; Ping, Li-Hua; Corti, Davide; Moldt, Brian; Hel, Zdenek; Lanzavecchia, Antonio; Ruprecht, Ruth M.; Burton, Dennis R.; Mestecky, Jiri; Anderson, Deborah J.; Forthal, Donald N.

2013-01-01

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure. PMID:24278022

SUPPRESSION OF THE EPITHELIAL-MESENCHYMAL TRANSITION BY GRAINYHEAD-LIKE-2

PubMed Central

Cieply, Benjamin; Riley, Philip; Pifer, Phillip M.; Widmeyer, Joseph; Addison, Joseph B.; Ivanov, Alexey V.; Denvir, James; Frisch, Steven M.

2012-01-01

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound healing processes and the cancer stem cell-like compartment of tumors, including TGF-β-dependence, we investigated the role of the Grainyhead gene, Grainyhead-Like-2 (GRHL2) in oncogenic EMT. GRHL2 was down-regulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis-sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir200b/c as well as the TGF-β receptor antagonist, BMP2. Lastly, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered a mesenchymal-to-epithelial transition and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells. PMID:22379025

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the "map" area, mainly in the basal epithelial cell layer. In "fingerprint" lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities.

Membrane Transport across Polarized Epithelia.

PubMed

Garcia-Castillo, Maria Daniela; Chinnapen, Daniel J-F; Lencer, Wayne I

2017-09-01

Polarized epithelial cells line diverse surfaces throughout the body forming selective barriers between the external environment and the internal milieu. To cross these epithelial barriers, large solutes and other cargoes must undergo transcytosis, an endocytic pathway unique to polarized cell types, and significant for the development of cell polarity, uptake of viral and bacterial pathogens, transepithelial signaling, and immunoglobulin transport. Here, we review recent advances in our knowledge of the transcytotic pathway for proteins and lipids. We also discuss briefly the promise of harnessing the molecules that undergo transcytosis as vehicles for clinical applications in drug delivery. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Human intraepithelial lymphocytes.

PubMed

Mayassi, Toufic; Jabri, Bana

2018-04-20

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ + CD8αβ + IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ + CD8αα + and adaptively induced TCRαβ + CD8αβ + IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.

USP17 is upregulated in osteosarcoma and promotes cell proliferation, metastasis, and epithelial-mesenchymal transition through stabilizing SMAD4.

PubMed

Song, Chenyang; Liu, Wenge; Li, Jiandong

2017-07-01

USP17 is upregulated in several cancers, indicating that USP17 might play essential functions in tumor development. However, the function of USP17 in osteosarcoma is still unknown. Our work aimed to investigate the function of USP17 in osteosarcoma. We found that the expression of USP17 was upregulated in osteosarcoma tissues and cell lines, including MG-63 and U2OS. Several functional experiments, such as colony formation analysis, Cell Counting Kit-8 assay, wound healing analysis, and transwell assay, showed that USP17 promoted cell proliferation, migration, and invasion. Moreover, we found that USP17 facilitated migration and invasion through promoting epithelial-mesenchymal transition. SMAD4 has been found to regulate epithelial-mesenchymal transition, co-immunopurification, and glutathione S-transferase pull-down analysis demonstrated that USP17 interacted with SMAD4. Furthermore, USP17 stabilized SMAD4 through its deubiquitinase activity. In conclusion, this study shows that USP17 enhances osteosarcoma cell proliferation and invasion through stabilizing SMAD4.

Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells

PubMed Central

Tam, Anthony; Wadsworth, Samuel; Dorscheid, Delbert; Man, Shu-Fan Paul; Sin, Don D.

2014-01-01

Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium. PMID:24964096

Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA.

PubMed

Vo, Nguyen T K; Bender, Aaron W; Ammendolia, Dustin A; Lumsden, John S; Dixon, Brian; Bols, Niels C

2015-07-01

A cell line, WE-spleen6, has been developed from the stromal layer of primary spleen cell cultures. On conventional plastic, WE-spleen6 cells had a spindle-shaped morphology at low cell density but grew to become epithelial-like at confluency. On the commercial extracellular matrix (ECM), Matrigel, the cells remained spindle-shaped and formed lumen-like structures. WE-spleen6 cells had intermediate filament protein, vimentin and the ECM protein, collagen I, but not smooth muscle α-actin (SMA) and von Willebrand factor (vWF) and lacked alkaline phosphatase and phagocytic activities. WE-spleen6 was more susceptible to infection with VHSV IVb than a fibroblast and epithelial cell lines from the walleye caudal fin, WE-cfin11f and WE-cfin11e, respectively. Viral transcripts and proteins appeared earlier in WE-spleen6 cultures as did cytopathic effect (CPE) and significant virus production. The synthetic double-stranded RNA (dsRNA), polyinosinic: polycytidylic acid (pIC), induced the antiviral protein Mx in both cell lines. Treating WE-spleen6 cultures with pIC prior to infection with VHSV IVb inhibited the early accumulation of viral transcripts and proteins and delayed the appearance of CPE and significant viral production. Of particular note, pIC caused the disappearance of viral P protein 2 days post infection. WE-spleen6 should be useful for investigating the impact of VHSV IVb on hematopoietic organs and the actions of pIC on the rhabdovirus life cycle. Copyright © 2015 Elsevier Ltd. All rights reserved.

Niclosamide inhibits epithelial-mesenchymal transition and tumor growth in lapatinib-resistant human epidermal growth factor receptor 2-positive breast cancer.

PubMed

Liu, Junjun; Chen, Xiaosong; Ward, Toby; Mao, Yan; Bockhorn, Jessica; Liu, Xiaofei; Wang, Gen; Pegram, Mark; Shen, Kunwei

2016-02-01

Acquired resistance to lapatinib, a human epidermal growth factor receptor 2 kinase inhibitor, remains a clinical problem for women with human epidermal growth factor receptor 2-positive advanced breast cancer, as metastasis is commonly observed in these patients. Niclosamide, an anti-helminthic agent, has recently been shown to exhibit cytotoxicity to tumor cells with stem-like characteristics. This study was designed to identify the mechanisms underlying lapatinib resistance and to determine whether niclosamide inhibits lapatinib resistance by reversing epithelial-mesenchymal transition. Here, two human epidermal growth factor receptor 2-positive breast cancer cell lines, SKBR3 and BT474, were exposed to increasing concentrations of lapatinib to establish lapatinib-resistant cultures. Lapatinib-resistant SKBR3 and BT474 cells exhibited up-regulation of the phenotypic epithelial-mesenchymal transition markers Snail, vimentin and α-smooth muscle actin, accompanied by activation of nuclear factor-кB and Src and a concomitant increase in stem cell marker expression (CD44(high)/CD24(low)), compared to naive lapatinib-sensitive SKBR3 and BT474 cells, respectively. Interestingly, niclosamide reversed epithelial-mesenchymal transition, induced apoptosis and inhibited cell growth by perturbing aberrant signaling pathway activation in lapatinib-resistant human epidermal growth factor receptor 2-positive cells. The ability of niclosamide to alleviate stem-like phenotype development and invasion was confirmed. Collectively, our results demonstrate that lapatinib resistance correlates with epithelial-mesenchymal transition and that niclosamide inhibits lapatinib-resistant cell viability and epithelial-mesenchymal transition. These findings suggest a role of niclosamide or derivatives optimized for more favorable bioavailability not only in reversing lapatinib resistance but also in reducing metastatic potential during the treatment of human epidermal growth factor receptor 2-positive breast cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

PubMed Central

Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

2013-01-01

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

IL-23 secreted by bronchial epithelial cells contributes to allergic sensitization in asthma model: role of IL-23 secreted by bronchial epithelial cells.

PubMed

Lee, Hyun Seung; Park, Da-Eun; Lee, Ji-Won; Chang, Yuna; Kim, Hye Young; Song, Woo-Jung; Kang, Hye-Ryun; Park, Heung-Woo; Chang, Yoon-Seok; Cho, Sang-Heon

2017-01-01

IL-23 has been postulated to be a critical mediator contributing to various inflammatory diseases. Dermatophagoides pteronyssinus (Der p) is one of the most common inhalant allergens. However, the role of IL-23 in Der p-induced mouse asthma model is not well understood, particularly with regard to the development of allergic sensitization in the airways. The objective of this study was to evaluate roles of IL-23 in Der p sensitization and asthma development. BALB/c mice were repeatedly administered Der p intranasally to develop Der p allergic sensitization and asthma. After Der p local administration, changes in IL-23 expression were examined in lung tissues and primary epithelial cells. Anti-IL-23p19 antibody was given during the Der p sensitization period, and its effects were examined. Effects of anti-IL-23p19 antibody at bronchial epithelial levels were also examined in vitro. The expression of IL-23 at bronchial epithelial layers was increased after Der p local administration in mouse. In Der p-induced mouse models, anti-IL-23p19 antibody treatment during allergen sensitization significantly diminished Der p allergic sensitization and several features of allergic asthma including the production of Th2 cytokines and the population of type 2 innate lymphoid cells in lungs. The activation of dendritic cells in lung-draining lymph nodes was also reduced by anti-IL-23 treatment. In murine lung alveolar type II-like epithelial cell line (MLE-12) cells, IL-23 blockade prevented cytokine responses to Der p stimulation, such as IL-1α, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-33, and also bone marrow-derived dendritic cell activation. In conclusion, IL-23 is another important bronchial epithelial cell-driven cytokine which may contribute to the development of house dust mite allergic sensitization and asthma. Copyright © 2017 the American Physiological Society.

Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

PubMed Central

Wang, Lihong; Cao, Hailong; Lu, Ning; Liu, Liping; Wang, Bangmao; Hu, Tianhui; Israel, Dawn A.; Peek, Richard M.; Polk, D. Brent; Yan, Fang

2013-01-01

Berberine, an isoquinoline alkaloid, is an active component of Ranunculaceae and Papaveraceae plant families. Berberine has been found to suppress growth of several tumor cell lines in vitro through the cell-type-dependent mechanism. Expression and activation of epidermal growth factor receptor (EGFR) is increased in colonic precancerous lesions and tumours, thus EGFR is considered a tumour promoter. The aim of this study was to investigate the effects and mechanisms of berberine on regulation of EGFR activity and proliferation in colonic tumor cell lines and in vivo. We reported that berberine significantly inhibited basal level and EGF-stimulated EGFR activation and proliferation in the immorto Min mouse colonic epithelial (IMCE) cells carrying the APC min mutation and human colonic carcinoma cell line, HT-29 cells. Berberine acted to inhibit proliferation through inducing G1/S and G2/M cell cycle arrest, which correlated with regulation of the checkpoint protein expression. In this study, we also showed that berberine stimulated ubiquitin ligase Cbl activation and Cbl's interaction with EGFR, and EGFR ubiquitinylation and down-regulation in these two cell lines in the presence or absence of EGF treatment. Knock-down Cbl expression blocked the effects of berberine on down-regulation of EGFR and inhibition of proliferation. Furthermore, berberine suppressed tumor growth in the HT-29 cell xenograft model. Cell proliferation and EGFR expression level was decreased by berberine treatment in this xenograft model and in colon epithelial cells of APC min/+ mice. Taken together, these data indicate that berberine enhances Cbl activity, resulting in down-regulation of EGFR expression and inhibition of proliferation in colon tumor cells. PMID:23457600

MicroRNA-9 enhances sensitivity to cetuximab in epithelial phenotype hepatocellular carcinoma cells through regulation of the eukaryotic translation initiation factor 5A-2.

PubMed

Xue, Fei; Liang, Yuntian; Li, Zhenrong; Liu, Yanhui; Zhang, Hongwei; Wen, Yu; Yan, Lei; Tang, Qiang; Xiao, Erhui; Zhang, Dongyi

2018-01-01

Hepatocellular carcinoma (HCC) is one of the most widespread malignant human tumors worldwide. Treatment options include radiotherapy, surgical intervention and chemotherapy; however, drug resistance is an ongoing treatment concern. In the present study, the effects of a microRNA (miR/miRNA), miR-9, on the sensitivity of HCC cell lines to the epidermal growth factor receptor inhibitor, cetuximab, were examined. miR-9 has been proposed to serve a role in tumorigenesis and tumor progression. In the present study, bioinformatics analyses identified the eukaryotic translation initiation factor 5A2 (eIF-5A-2) as a target of miR-9. The expression levels of miR-9 and eIF-5A-2 were examined by reverse transcription-quantitative polymerase chain reaction and HCC cell lines were transfected with miR-9 mimics and inhibitors to determine the effects of the miRNA on cell proliferation and viability. The miR-9 mimic was revealed to significantly increase the sensitivity of epithelial phenotype HCC cells (Hep3B and Huh7) to cetuximab, while the miR-9 inhibitor triggered the opposite effect. There were no significant differences in sensitivity to cetuximab observed in mesenchymal phenotype HCC cells (SNU387 and SNU449). Cells lines displaying high expression levels of eIF-5A-2 were more resistant to cetuximab. Transfection of cells with a miR-9 mimic resulted in downregulation of the expression of eIF-5A-2 mRNA, while an miR-9 inhibitor increased expression. When expression of eIF-5A-2 was knocked down with siRNA, the effects of miR-9 on cetuximab sensitivity were no longer observed. Taken together, these data support a role for miR-9 in enhancing the sensitivity of epithelial phenotype HCC cells to cetuximab through regulation of eIF-5A-2.

Human Urinary Epithelial Cells as a Source of Engraftable Hepatocyte-Like Cells Using Stem Cell Technology.

PubMed

Sauer, Vanessa; Tchaikovskaya, Tatyana; Wang, Xia; Li, Yanfeng; Zhang, Wei; Tar, Krisztina; Polgar, Zsuzsanna; Ding, Jianqiang; Guha, Chandan; Fox, Ira J; Roy-Chowdhury, Namita; Roy-Chowdhury, Jayanta

2016-12-13

Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.

DNA damage response at telomeres contributes to lung aging and chronic obstructive pulmonary disease

PubMed Central

Birch, Jodie; Anderson, Rhys K.; Correia-Melo, Clara; Jurk, Diana; Hewitt, Graeme; Marques, Francisco Madeira; Green, Nicola J.; Moisey, Elizabeth; Birrell, Mark A.; Belvisi, Maria G.; Black, Fiona; Taylor, John J.; Fisher, Andrew J.; De Soyza, Anthony

2015-01-01

Cellular senescence has been associated with the structural and functional decline observed during physiological lung aging and in chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the first line of defense in the lungs and are important to COPD pathogenesis. However, the mechanisms underlying airway epithelial cell senescence, and particularly the role of telomere dysfunction in this process, are poorly understood. We aimed to investigate telomere dysfunction in airway epithelial cells from patients with COPD, in the aging murine lung and following cigarette smoke exposure. We evaluated colocalization of γ-histone protein 2A.X and telomeres and telomere length in small airway epithelial cells from patients with COPD, during murine lung aging, and following cigarette smoke exposure in vivo and in vitro. We found that telomere-associated DNA damage foci increase in small airway epithelial cells from patients with COPD, without significant telomere shortening detected. With age, telomere-associated foci increase in small airway epithelial cells of the murine lung, which is accelerated by cigarette smoke exposure. Moreover, telomere-associated foci predict age-dependent emphysema, and late-generation Terc null mice, which harbor dysfunctional telomeres, show early-onset emphysema. We found that cigarette smoke accelerates telomere dysfunction via reactive oxygen species in vitro and may be associated with ataxia telangiectasia mutated-dependent secretion of inflammatory cytokines interleukin-6 and -8. We propose that telomeres are highly sensitive to cigarette smoke-induced damage, and telomere dysfunction may underlie decline of lung function observed during aging and in COPD. PMID:26386121